Methods and compositions for therapies using genes encoding secreted proteins such as interferon-beta

ABSTRACT

Methods and pharmaceutical compositions for modifying cells of a mammalian recipient with DNA encoding a secreted protein such as human interferon in situ are provided. The methods include forming a secreted protein expression system in vivo or ex vivo and administering the expression system to the mammalian recipient. The expression system and methods are useful for the localized and systemic delivery of interferons in situ.

This application is a continuation of U.S. application Ser. No.09/512,946, filed Feb. 25, 2000, now U.S. Pat. No. 6,696,423 (issued onFeb. 24, 2004), which is a continuation of PCT application numberPCT/US98/17606, filed 25 Aug. 1998, which claims benefit of UnitedStates provisional application No. 60/057,254, filed on 29 Aug. 1997.The disclosures of U.S. application Ser. No. 09/512,946, PCT applicationnumber PCT/US98/17606 and United States provisional application number60/057,254 are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to gene therapy. More specifically, the presentinvention relates to delivery of DNA encoding secreted proteins such asinterferon proteins in humans and animals.

BACKGROUND OF THE INVENTION

Interferons (also referred to as “IFN” or “IFNs”) are proteins having avariety of biological activities, some of which are antiviral,immunomodulating and antiproliferative. They are relatively small,species-specific, single chain polypeptides, produced by mammalian cellsin response to exposure to a variety of inducers such as viruses,polypeptides, mitogens and the like. Interferons protect animal tissuesand cells against viral attack and are an important host defensemechanism. In most cases, interferons provide better protection totissues and cells of the kind from which they have been produced than toother types of tissues and cells, indicating that human-derivedinterferon could be more efficacious in treating human diseases than interferons from other species.

There are several distinct types of human interferons, generallyclassified as leukocyte (interferon-alpha [α]), fibroblast(interferon-beta [β]) and immune (interferon-gamma [γ]), and a largenumber of variants thereof. General discussions of interferons can befound in various texts and monographs including: The Interferon System(W. E. Stewart, II, Springer-Verlag, N.Y. 1979); and Interferon Therapy(World Health Organization Technical Reports Series 676, World HealthOrganization, Geneva 1982), incorporated herein by reference.

Interferons have potential in the treatment of a large number of humancancers since these molecules have anti-cancer activity which acts atmultiple levels. First, interferon proteins can directly inhibit theproliferation of human tumor cells. The anti-proliferative activity isalso synergistic with a variety of approved chemotherapeutic agents suchas cis-platin, 5FU and taxol. Secondly, the immunomodulatory activity ofinterferon proteins can lead to the induction of an anti-tumor immuneresponse. This response includes activation of NK cells, stimulation ofmacrophage activity and induction of MHC class I surface expressionleading to the induction of anti-tumor cytotoxic T lymphocyte activity.Moreover, some studies further indicate that IFN-β protein may haveanti-angiogenic activity. Angiogenesis, new blood vessel formation, iscritical for the growth of solid tumors. Evidence indicates that IFN-βmay inhibit angiogenesis by inhibiting the expression of pro-angiogenicfactors such as bFGF and VEGF. Lastly, interferon proteins may inhibittumor invasiveness by affecting the expression of enzymes such ascollagenase and elastase which are important in tissue remodeling.

Interferons also appear to have antiviral activities that are based ontwo different mechanisms. For instance, type I interferon proteins (αand β) can directly inhibit the replication of human hepatitis B virus(“HBV”) and hepatitis C virus (“HCV”), but can also stimulate an immuneresponse which attacks cells infected with these viruses.

Specifically, and despite its potential therapeutic value, interferonproteins have only had limited clinical success against viral hepatitisand solid tumors. IFN-α has been approved for the treatment of both HBVand HCV; however, the response rate in both cases is only approximately20%. While interferon proteins have been approved for the treatment ofsome cancers such as lymphomas, leukemias, melanoma and renal cellcarcinoma, the majority of clinical trials in which interferons are usedalone or in combination with conventional chemotherapeutic agents in thetreatment of solid tumors have been unsuccessful.

The method of administering interferon is an important factor in theclinical application of this important therapeutic agent. Systemicadministration of interferon protein by either intravenous,intramuscular or subcutaneous injection has been most frequently usedwith some success in treating disorders such as hairy cell leukemia,Acquired Immune Deficiency Syndrome (AIDS) and related Kaposi's sarcoma.It is known, however, that proteins in their purified form areespecially susceptible to degradation. In particular, forinterferon-beta, the primary mechanism(s) of interferon degradation insolution are aggregation and deamidation. The lack of interferonstability in solutions and other products has heretofore limited itsutility. Furthermore, following parenteral interferon proteinadministration (intramuscular, subcutaneous or intravenous) theclearance rate of interferon protein is very rapid. Therefore,parenteral protein administration may not allow the localization ofsufficient interferon at the active site (the solid tumor, or, in thecase of hepatitis, the liver). The amount of interferon that can begiven parenterally in patients is limited by the side-effects observedat high interferon doses. A more effective therapy is clearly needed.

SUMMARY OF THE INVENTION

This application is directed toward eliminating the problems associatedwith delivering a secreted protein such as interferon protein as atherapeutic. The present invention is directed to a method of interferontherapy in which the gene encoding the secreted protein rather than theprotein itself, is delivered.

Accordingly, it is one object of the instant invention to provide amethod of gene therapy based on the use of genetically engineered cellsand to the use thereof for delivering a secreted protein such as aninterferon to a mammalian recipient. The instant invention satisfiesthese and other objects by providing methods for forming a cellexpression system, the expression system produced thereby andpharmaceutical compositions containing the same. The cell expressionsystem expresses a gene encoding one or more secreted proteins and isuseful as a vehicle for delivering the gene product to the mammalianrecipient in situ. In a preferred embodiment, the mammalian recipient isa human.

In one embodiment of the invention, a cell expression system isdescribed for expressing in a cell of a mammalian recipient in vivo, aninterferon protein for treating a condition. The expression systemcomprises a cell of the same species as the mammalian recipient and anexpression vector contained therein for expressing the interferonprotein. Preferably, the mammalian recipient is a human and theexpression vector comprises a viral vector.

In another embodiment, the expression system comprises a plurality ofcells of the same species as the mammalian recipient and an expressionvector contained therein for expressing the secreted protein. Theexpression vector is contained within only a portion of the plurality ofcells. Preferably, at least 0.3% by number of the cells contain thevector. The preferred secreted protein is an interferon and the mostpreferred interferons are alpha, beta, gamma and consensus interferon,with beta interferon being the most preferred.

In other embodiments, the cell expression system comprises a pluralityof cancer cells and at least a portion of the cancer cells contain anadenoviral vector having an isolated polynucleotide encoding, uponexpression, an interferon. In this cell expression system, theadenoviral vectors are selected from the group consisting of: (a) anadenoviral vector having a deletion and/or mutation in its E1 gene; (b)an adenoviral vector having a deletion and/or mutation in its E2a gene,said vector expressing human interferon-beta; (c) an adenoviral vectorhaving a deletion and/or mutation in both its E1 and E4 genes, and (d)an adenoviral vector having a deletion of all of its genes; said vectorexpressing human interferon-beta.

A pharmaceutical composition for delivery of a secreted protein to asite of a mammalian recipient, is also encompassed within the invention.The composition comprises a carrier and a plurality of geneticallymodified cells of the same species as the mammalian recipient and atleast a portion of the cells contain an expression vector for expressingan effective amount of the secreted protein. The preferred secretedprotein is an interferon. The composition encompasses compositions forboth in vivo and ex vivo delivery.

A method for making an ex vivo gene therapy pharmaceutical preparationfor administration to a mammalian recipient is another embodiment. Themethod includes the steps of: (a) forming a plurality of cells of thesame species as the mammalian recipient, (b) introducing an expressionvector for expressing a secreted protein into at least one cell of theplurality to form at least one genetically modified cell and (c) placingthe at least one genetically modified cell in a pharmaceuticallyacceptable carrier to obtain a pharmaceutical preparation that issuitable for administration to a site of the mammalian recipient.

Another embodiment of the invention is a method for gene therapy, whichcomprises genetically modifying at least one cell of a mammalianrecipient via the steps of (a) introducing an expression vector forexpressing a secreted protein into at least one cell to form at leastone genetically modified cell and (b) allowing the genetically modifiedcell to contact a site of the mammalian recipient. This method mayfurther comprise the step of removing at least one cell from themammalian recipient prior to the step of introducing the expressionvector. In yet another embodiment, the step of introducing the vectorcomprises introducing a vector to only a portion of the plurality ofcells.

A method of ex vivo gene therapy is encompassed by the invention andincludes the steps of removing a plurality of cells from a subject;administering a recombinant adenovirus to at least one cell of theplurality of cells, such that there exists an excess of cells notcontaining the adenovirus. The adenovirus can have a deletion in its E1gene and includes an isolated polynucleotide encoding a secretedprotein. The plurality of cells is reintroduced back into the subject.

In a method of in vivo gene therapy, the steps include administering anadenoviral vector that includes an isolated polynucleotide encodinghuman interferon-beta (β) protein directly into a cell of a subjectwithout first removing said cell from the subject. The in vivo and exvivo methods allow for topical, intraocular, parenteral, intranasal,intratracheal, intrabronchial, intramuscular, subcutaneous, intravenous,and intraperitoneal administration.

The present invention has several advantages. Interferon gene therapymay allow for very high local interferon concentrations with lowsystemic levels. This could result in greater efficacy with lowerside-effects. Also, interferons delivered by gene therapy would bepresent at a fairly constant level, unlike the situation observed ininterferon protein therapy in which very high interferon “bursts” orpeaks in protein concentration (which could lead to toxicity) that occurafter protein injection, are followed by very low levels in which theinterferon concentration is likely to be too low to be effective.

Patient convenience is also a critical factor. While frequent injectionsof interferon protein are necessary, a single administration, or a fewinfrequent administrations, of a vector expressing the interferon genecould provide long-term stable production of the protein. Gene therapycould allow the delivery of interferons in a controlled manner to adistinct target organ or tumor. An autocrine system can be establishedin which the same cells express, secrete and take up the interferon.Thus, very high local doses can be achieved. These high doses cannot beachieved by parenteral protein administration due to toxicity problems.

Since interferon proteins are secreted out of cells, every cell in atumor or in the hepatitis-infected liver need not be transduced by theinterferon gene. Those cells which do not take up the gene will beaffected by neighboring cells (the so-called “bystander effect”) whichhave the gene and secrete the interferon protein. This is a significantfinding and is likely to have dramatic effects on treatment regimenssince not every cell in a tumor mass or in an organ need contain anexpression vector.

Lastly, parenteral interferon administration has been shown to lead tothe generation of anti-interferon antibodies. It may be that thispotentially neutralizing antibody response can be lessened followingintroduction of the interferon gene to a distinct local region. Besidesthe local expression, the interferon expressed will be produced byendogenous human cells and, therefore, will be more natural in itsstructure and glycosylation and, possibly, less immunogenic thaninterferon protein produced in bacteria, yeast or Chinese hamster ovarycells and then purified and injected parenterally.

These and other aspects of the invention as well as various advantagesand utilities will be more apparent with reference to the detaileddescription of the preferred embodiments and to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. (A) Either uninfected MDA-MB-468 (-∘-) or cells infected withH5.110hIFNβ cells at 0.01% (-Δ-), 0.03% (-□-), 0.1% (-∇-), or 0.3% (-

-) were injected subcutaneously into the flanks of nude mice and meantumor size was plotted versus the time following tumor cell implantation(B) Kaplan-Meir plot showing the percentage survival of mice over theobservation period of 109 days. Uninfected cells (-∘-); H5.100hIFNβinfected cells at 0.01% (-Δ-); 0.03% (-□-), 0.1% (-∇-); 0.3% (-

-).

FIG. 2. (A) Ex vivo interferon beta gene therapy in: (1) KM12L4A cells;(2) Huh7 cells; and (3) ME180 cells. Mean tumor size was plotted versustime following tumor cell implantation. Mice were implanted withuninfected cells (-▪-), or H5.110hIFNβ infected cells at 1% (-●-), or10% (-▴-). In those groups in which some mice were sacrificed, the tumorsize is presented as the average with the last value carried forward forthe sacrificed animals. Discontinuation of the plots reflects the deathor sacrifice of all animals in a group. (B) Percentage survival of miceover the observation period of 70 days. Panels 1, 2, and 3 show the datagenerated with mice implanted with KM12L4A cells, Huh7 cells, and ME180cells respectively. The symbols represent mice that were implanted withuninfected cells (-▪-), H5.110hIFNβ infected cells at 1% (-●-) or 10%(-▴-).

FIG. 3. Direct in vivo treatment of established MDA-MB-468 tumors.Tumors were injected with H5.110hIFNβ at 3×10⁹ pfu (-

-), 1×10⁹ pfu (-

-), 3×10⁸ pfu (-∘-), 1×10⁸ pfu (-Δ-), and 3×10⁷ pfu (-∇-) respectively,or with PBS (-

-), or with H5.110lacZ at 3×10⁹ pfu (-⋄-), 1×10⁹ pfu (-▴-), 3×10⁸ pfu(-×-), and 1×10⁸ pfu (-

-) respectively. Tumor sizes were measured over a period of 14 daysfollowing the treatment injections.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based, in part, on development of an ex vivogene therapy method that uses a cell that (1) can be easily removed fromthe patient, (2) can be modified in vitro by introduction of geneticmaterial; (3) can be conveniently implanted in the recipient; (4) isnon-thrombogenic; and (5) can be implanted into the recipient in largenumbers. The disclosed in vivo gene therapy method uses a cell that (1)is present in the recipient and (2) can be modified in situ to expressisolated genetic material. The genetically modified cell includesregulatory elements for controlling the amount of genetic materialexpressed.

The genetically modified cells will survive and continue to produce theexpressed material in situ for an amount of time necessary for theexpressed material to have a beneficial (i.e., therapeutic) effect,without interfering with the normal function of the tissue in which thecells are located.

Preferably, the expressed material is a secreted protein (definedbelow). Most preferably, the secreted protein is an interferon. Indeed,although interferons are the most preferred therapeutic agent, a generalprinciple applicable to gene therapy with any secreted protein has beenfound. We have found that, due to the fact that secreted proteins suchas interferons are released from the cells in which they have beenexpressed, every cell in a tumor mass or in, for instance, ahepatitis-infected liver need not be transduced by the “secretedprotein” gene. Those cells which do not take up the gene will beaffected by neighboring cells which have the gene and secrete theprotein (i.e., the so-called “bystander effect”). Although gene therapyis described with isolated polynucleotides that encode, upon expression,for interferons, any secreted protein (as defined below) has potentialin the present method and compositions.

All citations recited in this document are incorporated herein byreference.

I. Definitions:

“Gene therapy”—a procedure in which a disease phenotype is correctedthrough the introduction of genetic information into the affectedorganism.

“ex vivo gene therapy”—a procedure in which cells are removed from asubject and cultured in vitro. A polynucleotide such as a functionalgene is introduced into the cells in vitro, the modified cells areexpanded in culture, and then reimplanted in the subject.

“in vivo gene therapy”—a procedure in which target cells are not removedfrom the subject. Rather, the transferred polynucleotide (e.g., a geneencoding, upon expression, for an interferon) is introduced into cellsof the recipient organism in situ, that is, within the recipient. Invivo gene therapy has been examined in several animal models and recentpublications have reported the feasibility of direct gene transfer insitu into organs and tissues.

“condition amenable to gene therapy”—embraces conditions such as geneticdiseases (i.e., a disease condition that is attributable to one or moregene defects), acquired pathologies (i.e., a pathological conditionwhich is not attributable to an inborn defect), cancers and prophylacticprocesses (i.e., prevention of a disease or of an undesired medicalcondition).

“acquired pathology”—refers to a disease or syndrome manifested by anabnormal physiological, biochemical, cellular, structural, or molecularbiological state.

“polynucleotide”—a polymer of nucleic acid monomeric units, themonomeric units being either ribonucleic acids (RNA), deoxyribonucleicacids (DNA), or combinations of both. The four DNA bases are adenine(A), guanine (G), cytosine (C) and thymine (T). The four RNA bases areA, G, C, and uracil (U).

“isolated”—when applied to polynucleotide sequences of genes that encodean interferon, means an RNA or DNA polynucleotide, portion of genomicpolynucleotide, cDNA or synthetic polynucleotide which, by virtue of itsorigin or manipulation: (i) is not associated with all of apolynucleotide with which it is associated in nature (e.g., is presentin a host cell as a portion of an expression vector); or (ii) is linkedto a nucleic acid or other chemical moiety other than that to which itis linked in nature; or (iii) does not occur in nature. By “isolated” itis further meant a polynucleotide sequence: (i) amplified in vitro by,for example, polymerase chain reaction (PCR); (ii) chemicallysynthesized; (iii) recombinantly produced by cloning; or (iv) purified,as by cleavage and gel separation.

Thus, an “isolated” interferon polynucleotide includes, for example, anon-naturally occurring nucleic acid that can be transcribed intoanti-sense RNA, as well as a gene encoding an interferon protein whichis not expressed or is expressed at biologically insignificant levels ina naturally-occurring cell. To illustrate, a synthetic or natural geneencoding human interferon-beta 1a would be considered “isolated” withrespect to human brain cells since the latter cells do not naturallyexpress interferon-beta 1a. Still another example of an “isolatedpolynucleotide” is the introduction of only part of an interferon geneto create a recombinant gene, such as combining an inducible promoterwith an endogenous interferon coding sequence via homologousrecombination.

“gene”—a DNA sequence (i.e., a linear array of nucleotides connected toeach other by 3′-5′ pentose phosphodiester bonds) which encodes throughits mRNA an amino acid sequence of a specific protein.

“transcription”—the process of producing mRNA from a gene.

“translation”—the process of producing a protein from mRNA.

“expression”—the process undergone by a DNA sequence or a gene toproduce a protein, combining transcription and translation.

“inhibiting growth”—as used herein this term refers to both theinhibition of target cell (i.e., tumor) growth and inhibition of thetransformed phenotype (as measured by, for example, changes inmorphology).

“amino acid”—a monomeric unit of a peptide, polypeptide, or protein.There are twenty L-isomers of amino acids. The term also includesanalogs of the amino acids and D-isomers of the protein amino acids andtheir analogs.

“protein”—any polymer consisting essentially of any of the 20 proteinamino acids, regardless of its size. Although “polypeptide” is oftenused in reference to relatively large polypeptides, and “peptide” isoften used in reference to small polypeptides, usage of these terms inthe art overlaps and is varied. The term “protein” as used herein refersto peptides, proteins and polypeptides, unless otherwise noted.

“secreted protein”—a protein which is transported from the inside of acell to the exterior of the cell; among secreted proteins are a largenumber of growth factors and immunomodulator proteins such as thevarious interferons (α, β, γ), interleukins such as IL-1, −2, −4, −8,and −12 and growth factors such as GM-CSF, G-CSF.

“genetic fusion”—refers to a co-linear, covalent linkage of two or moreproteins via their individual peptide backbones, through geneticexpression of a polynucleotide molecule encoding those proteins.

“mutant”—any change in quality or structure of genetic material of anorganism, in particular any change (i.e., deletion, substitution,addition, or alteration) in a wild type interferon gene or any change ina wild type interferon protein.

“wild type”—the naturally-occurring polynucleotide or amino acidsequence of an interferon gene or interferon protein, respectively, asit exists in vivo.

“standard hybridization conditions”—salt and temperature conditionssubstantially equivalent to 0.5×SSC to about 5×SSC and 65 degrees C. forboth hybridization and wash. The term “standard hybridizationconditions” as used herein is therefore an operational definition andencompasses a range of hybridizations. Higher stringency conditions may,for example, include hybridizing with plaque screen buffer (0.2%polyvinylpyrrolidone, 0.2% Ficoll 400; 0.2% bovine serum albumin, 50 mMTris-HCl (pH 7.5); 1 M NaCl; 0.1% sodium pyrophosphate; 1% SDS); 10%dextran sulphate, and 100 ug/ml denatured, sonicated salmon sperm DNA at65 degrees C. for 12-20 hours, and washing with 75 mM NaCl/7.5 mM sodiumcitrate (0.5×SSC)/1% SDS at 65 degrees C. Lower stringency conditionsmay, for example, include hybridizing with plaque screen buffer, 10%dextran sulphate and 110 ug/ml denatured, sonicated salmon sperm DNA at55 degrees C. for 12-20 hours, and washing with 300 mM NaCl/30 mM sodiumcitrate (2.0×SSC)/1% SDS at 55 degrees C.

“expression control sequence”—a sequence of nucleotides that controlsand regulates expression of genes when operatively linked to thosegenes.

“operatively linked”—a polynucleotide sequence (DNA, RNA) is operativelylinked to an expression control sequence when the expression controlsequence controls and regulates the transcription and translation ofthat polynucleotide sequence. The term “operatively linked” includeshaving an appropriate start signal (e.g., ATG) in front of thepolynucleotide sequence to be expressed and maintaining the correctreading frame to permit expression of the polynucleotide sequence underthe control of the expression control sequence and production of thedesired interferon encoded by the isolated polynucleotide sequence.

“expression vector”—a polynucleotide, most commonly a DNA plasmid (butwhich also includes a virus) which allows expression of at least onegene when the expression vector is introduced into a host cell. Thevector may, or may not, be able to replicate in a cell.

“tumor”—any undesirable proliferation of cells. Such growth includesmalignant and non-malignant, solid or fluid tumors, carcinomas,myelomas, sarcomas, leukemias, lymphomas and other cancerous, neoplasticor tumorigenic diseases.

“genetically modified cell” (also called a “cell expressionsystem”)—comprises a cell and an expression vector for expressing theinterferon protein. For ex vivo purposes, the genetically modified cellsare suitable for administration to a mammalian recipient, where theyreplace or co-exist with the endogenous cells of the recipient. For invivo purposes, the cells are created inside the recipient.

The instant invention also provides various methods for making and usingthe above-described genetically-modified cells. In particular, theinvention provides a method for genetically modifying cell(s) of amammalian recipient ex vivo and administering the genetically modifiedcells to the mammalian recipient. In a preferred embodiment for ex vivogene therapy, the cells are autologous cells, i.e., cells removed fromthe mammalian recipient. As used herein, the term “removed” means a cellor a plurality of cells that have been removed from theirnaturally-occurring in vivo location. Methods for removing cells from apatient, as well as methods for maintaining the isolated cells inculture are known to those of ordinary skill in the art (Stylianou, E.,et al., Kidney Intl. 37:1563-1570 (1992); Hjelle, J. H., et al.,Peritoneal Dialysis Intl. 9: 341-347 (1989); Heldin, P. Biochem. J. 283:165-170 (1992); Di Paolo, N., et al., Int. J. Art. Org. 12: 485-501(1989); Di Paolo, N., et al., Clinical Nephrol. 34: 179-184 (1990); DiPaolo, N., et al., Nephron 57: 323-331 (1991)). All patents, patentapplications and publications mentioned in the Detailed Description ofthe Invention, herein, both supra and infra, are incorporated herein byreference. ps II. Isolated, Interferon Polynucleotides

An “interferon” (also referred to as “IFN”) is a small,species-specific, single chain polypeptide, produced by mammalian cellsin response to exposure to a variety of inducers such as viruses,polypeptides, mitogens and the like. The most preferred interferons arein recombinant form and recombinant DNA methods for producing proteinsincluding the various interferons are known and are not intended tolimit the invention in any way. See for example, U.S. Pat. Nos.4,399,216, 5,149,636, 5,179,017 (Axel et al) and 4,470,461 (Kaufman).Recombinant forms of interferon-alpha, beta, gamma and consensusinterferon have been produced. Forms of interferon may be expressed fromcells containing polynucleotide sequences encoding variants such ascysteine-depleted mutants (e.g., for interferon-beta) andmethionine-depleted mutants. Other modifications may take place throughthe post-translational processing systems of the host cell. The exactchemical structure of a particular interferon will therefore depend onseveral factors and is not intended to limit the scope of the invention.All such interferon proteins included in the formulations describedherein will retain their bioactivity when placed in suitableenvironmental conditions.

Preferred polynucleotides that may be used in the present methods of theinvention are derived from the wild-type interferon gene sequences ofvarious vertebrates, preferably mammals and are obtained using methodsthat are well-known to those having ordinary skill in the art. See, forexample: U.S. Pat. No. 5,641,656 (issued Jun. 24, 1997: DNA encodingavian type I interferon proprotein and mature avian type I interferon),U.S. Pat. No. 5,605,688 (Feb. 25, 1997—recombinant dog and horse type Iinterferons); U.S. Pat. No. 5,554,513 (Sep. 10, 1996; DNA sequence whichcodes for human interferon-beta2A); U.S. Pat. No. 5,541,312; Jul. 30,1996—DNA which codes for human fibroblast beta -2 interferonpolypeptide); U.S. Pat. No. 5,231,176 (Jul. 27, 1993, DNA moleculeencoding a human leukocyte interferon); ); U.S. Pat. No. 5,071,761 (Dec.10, 1991, DNA sequence coding for sub-sequences of human lymphoblastoidinterferons LyIFN-alpha-2 and LyIFN-alpha -3); U.S. Pat. No. 4,970,161(Nov. 13, 1990, DNA sequence coding for human interferon-gamma); U.S.Pat. No. 4,738,931 (Apr. 19, 1988, DNA containing a human interferonbeta gene); U.S. Pat. No. 4,695,543 (Sep. 22, 1987, humanalpha-interferon Gx-1 gene and U.S. Pat. No. 4,456,748 (Jun. 26, 1984,DNA encoding sub-sequences of different, naturally, occurring leukocyteinterferons).

Mutant members of the interferon family of genes may be used inaccordance with this invention. Mutations in the wild-type interferonpolynucleotide sequence are developed using conventional methods ofdirected mutagenesis, known to those of ordinary skill in the art. Theterm “mutant” is also meant to encompass genetic fusions so that thefollowing interferon sequences, incorporated herein by reference, wouldall be considered “mutant” sequences:

U.S. Pat. No. 5,273,889 (Dec. 28, 1993, DNA construct comprisinggamma-interferon gene linked to a sequence encoding an immunogenicleukotoxin); U.S. Pat. No. 4,959,314 (Sep. 25, 1990, Gene having a DNAsequence that encodes a synthetic mutein of a biologically active nativeprotein); U.S. Pat. No. 4,929,554 (May 29, 1990, DNA encodingdes-CYS-TYR-CYS recombinant human immune interferon); U.S. Pat. No.4,914,033 (Apr. 3, 1990, DNA molecule encoding a modified betainterferon comprising a beta interferon); and U.S. Pat. No. 4,569,908(Feb. 11, 1986, DNA having a nucleotide sequence that encodes amulticlass hybrid interferon polypeptide).

Moreover, the isolated polynucleotides described in these patents can bealtered to provide for functionally equivalent polynucleotides. Apolynucleotide is “functionally equivalent” compared with those of theabove sequences if it satisfies at least one of the followingconditions:

-   -   (a): the “functional equivalent” is a polynucleotide that        hybridizes to any of the foregoing sequences under standard        hybridization conditions and/or is degenerate to any of the        foregoing sequences. Most preferably, it encodes a mutant        interferon having the therapeutic activity of a wild type        interferon;    -   (b) the “functional equivalent” is a polynucleotide that codes        on expression for an amino acid sequence encoded by any of the        polynucleotides of the foregoing interferon sequences.

In summary, the term “interferon” includes, but is not limited to, theagents listed above as well as their functional equivalents. As usedherein, the term “functional equivalent” therefore refers to aninterferon protein or a polynucleotide encoding the interferon proteinthat has the same or an improved beneficial effect on the mammalianrecipient as the interferon of which is it deemed a functionalequivalent. As will be appreciated by one of ordinary skill in the art,a functionally equivalent protein can be produced by recombinanttechniques, e.g., by expressing a “functionally equivalent DNA”.Accordingly, the instant invention embraces interferons encoded bynaturally-occurring DNAs, as well as by non-naturally-occurring DNAswhich encode the same protein as encoded by the naturally-occurring DNA.Due to the degeneracy of the nucleotide coding sequences, otherpolynucleotides may be used to encode interferons. These include all, orportions of the above sequences which are altered by the substitution ofdifferent codons that encode the same amino acid residue within thesequence, thus producing a silent change. Such altered sequences areregarded as equivalents of these sequences. For example, Phe (F) iscoded for by two codons, TTC or TTT, Tyr (Y) is coded for by TAC or TATand His (H) is coded for by CAC or CAT. On the other hand, Trp (W) iscoded for by a single codon, TGG. Accordingly, it will be appreciatedthat for a given DNA sequence encoding a particular interferon therewill be many DNA degenerate sequences that will code for it. Thesedegenerate DNA sequences are considered within the scope of thisinvention.

Consensus interferon is also included within this definition. Asemployed herein, “consensus interferon” is a nonnaturally occurringpolypeptide, which predominantly includes those amino acid residues thatare common to all naturally-occurring human interferon subtype sequencesand which include, at one or more of those positions where there is noamino acid common to all subtypes, an amino acid which predominantlyoccurs at that position and in no event includes any amino acid residuewhich is not extant in that position in at least one naturally-occurringsubtype. Consensus interferon sequences encompass consensus sequences ofany of the above-referenced interferons provided that they have subtypesequences. Exemplary consensus interferons are disclosed in U.S. Pat.Nos. 4,695,623 and 4,897,471 (Amgen). DNA sequences encoding consensusinterferon may be synthesized as described in these patents or by otherstandard methods. Consensus interferon polypeptides are preferably theproducts of expression of manufactured DNA sequences, transformed ortransfected into hosts, as described herein. That is, consensusinterferon is preferably recombinantly produced. Such materials may bepurified by procedures well known in the art.

The above-disclosed interferons and conditions amenable to genereplacement therapy are merely illustrative and are not intended tolimit the scope of the instant invention. The selection of a suitableinterferon for treating a known condition is deemed to be within thescope of one of ordinary skill of the art without undue experimentation.

III. Methods for Introducing Polynucleotide Sequences of SecretedProteins into Cells

The term “transformation” or “transform” refers to any geneticmodification of cells and includes both “transfection” and“transduction”.

As used herein, “transfection of cells” refers to the acquisition by acell of new genetic material by incorporation of added DNA. Thus,transfection refers to the insertion of nucleic acid (e.g., DNA) into acell using physical or chemical methods. Several transfection techniquesare known to those of ordinary skill in the art including: calciumphosphate DNA co-precipitation (Methods in Molecular Biology, Vol. 7,Gene Transfer and Expression Protocols, Ed. E. J. Murray, Humana Press(1991)); DEAE-dextran (supra); electroporation (supra); cationicliposome-mediated transfection (supra); and tungstenparticle-facilitated microparticle bombardment (Johnston, S. A., Nature346: 776-777 (1990)); and strontium phosphate DNA co-precipitation(Brash D. E. et al. Molec. Cell. Biol. 7: 2031-2034 (1987). Each ofthese methods is well represented in the art.

In contrast, “transduction of cells” refers to the process oftransferring nucleic acid into a cell using a DNA or RNA virus. One ormore isolated polynucleotide sequences encoding one or more interferonproteins contained within the virus may be incorporated into thechromosome of the transduced cell. Alternatively, a cell is transducedwith a virus but the cell will not have the isolated polynucleotideincorporated into its chromosomes but will be capable of expressinginterferon extrachromosomally within the cell.

According to one embodiment, the cells are transformed (i.e.,genetically modified) ex vivo. The cells are isolated from a mammal(preferably a human) and transformed (i.e., transduced or transfected invitro) with a vector containing an isolated polynucleotide such as arecombinant gene operatively linked to one or more expression controlsequences for expressing a recombinant secreted protein (e.g., aninterferon). The cells are then administered to a mammalian recipientfor delivery of the protein in situ. Preferably, the mammalian recipientis a human and the cells to be modified are autologous cells, i.e., thecells are isolated from the mammalian recipient. The isolation andculture of cells in vitro has been reported

According to another embodiment, the cells are transformed or otherwisegenetically modified in vivo. The cells from the mammalian recipient(preferably a human), are transformed (i.e., transduced or transfected)in vivo with a vector containing isolated polynucleotide such as arecombinant gene operatively linked to one or more expression controlsequences for expressing a secreted protein (i.e., recombinantinterferon) and the protein is delivered in situ.

The isolated polynucleotides encoding the secreted protein (e.g., a cDNAencoding one or more therapeutic interferon proteins) is introduced intothe cell ex vivo or in vivo by genetic transfer methods, such astransfection or transduction, to provide a genetically modified cell.Various expression vectors (i.e., vehicles for facilitating delivery ofthe isolated polynucleotide into a target cell) are known to one ofordinary skill in the art.

Typically, the introduced genetic material includes an isolatedpolynucleotide such as an interferon gene (usually in the form of a cDNAcomprising the exons coding for the interferon) together with a promoterto control transcription of the new gene. The promotercharacteristically has a specific nucleotide sequence necessary toinitiate transcription. Optionally, the genetic material could includeintronic sequences which will be removed from the mature transcript byRNA splicing. A polyadenylation signal should be present at the 3′ endof the gene to be expressed. The introduced genetic material also mayinclude an appropriate secretion “signal” sequence for secreting thetherapeutic gene product (i.e., an interferon) from the cell to theextracellular milieu.

Optionally, the isolated genetic material further includes additionalsequences (i.e., enhancers) required to obtain the desired genetranscription activity. For the purpose of this discussion an “enhancer”is simply any non-translated DNA sequence which works contiguous withthe coding sequence (in cis) to change the basal transcription leveldictated by the promoter.

Preferably, the isolated genetic material is introduced into the cellgenome immediately downstream from the promoter so that the promoter andcoding sequence are operatively linked so as to permit transcription ofthe coding sequence. Preferred viral expression vectors include anexogenous promoter element to control transcription of the insertedinterferon gene. Such exogenous promoters include both constitutive andinducible promoters.

Naturally-occurring constitutive promoters control the expression ofproteins that regulate essential cell functions. As a result, a geneunder the control of a constitutive promoter is expressed under allconditions of cell growth. Exemplary constitutive promoters include thepromoters for the following genes which encode certain constitutive or“housekeeping” functions: hypoxanthine phosphoribosyl transferase(HPRT), dihydrofolate reductase (DHFR) (Scharfmann et al., Proc. Natl.Acad. Sci. USA 88: 4626-4630 (1991)), adenosine deaminase,phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase,the β-actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86:10006-10010 (1989)), and other constitutive promoters known to those ofskill in the art.

In addition, many viral promoters function constitutively in eucaryoticcells. These include: the early and late promoters of SV40 (See Bernoistand Chambon, Nature, 290:304 (1981)); the long terminal repeats (LTRs)of Moloney Leukemia Virus and other retroviruses (See Weiss et al., RNATumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1985)); the thymidine-kinase promoter of Herpes Simplex Virus (HSV)(See Wagner et al., Proc. Nat. Acad. Sci. USA, 78: 1441 (1981)); thecytomegalovirus immediate-early (IE1) promoter (See Karasuyama et al.,J. Exp. Med., 169: 13 (1989); the promoter of the Rous sarcoma virus(RSV) (Yamamoto et al., Cell, 22:787 (1980)); the adenovirus major latepromoter (Yamada et al., Proc. Nat. Acad. Sci. USA, 82: 3567 (1985)),among many others. Accordingly, any of the above-referenced constitutivepromoters can be used to control transcription of a gene insert. (Seealso Section B).

If delivery of the interferon gene is to specific tissues, it may bedesirable to target the expression of this gene. For instance, there aremany promoters described in the literature which are only expressed incertain tissues. Examples include liver-specific promoters of hepatitisB virus (Sandig et al., Gene Therapy 3: 1002-1009 (1996) and the albumingene (Pinkert et al., Genes and Development, 1: 268-276 (1987); see alsoGuo et al., Gene Therapy, 3: 802-810 (1996) for other liver-specificpromoter. Moreover, there are many promoters described in the literaturewhich are only expressed in specific tumors. Examples include the PSApromoter (prostate carcinoma), carcinoembryonic antigen promoter (colonand lung carcinoma), β-casein promoter (mammary carcinoma), tyrosinasepromoter (melanoma), calcineurin Aα promoter (glioma, neuroblastoma),c-sis promoter (osteosarcoma) and the α-fetoprotein promoter (hepatoma).

Genes that are under the control of inducible promoters are expressedonly, or to a greater degree, in the presence of an inducing agent,(e.g., transcription under control of the metallothionein promoter isgreatly increased in presence of certain metal ions). See also theglucocorticoid-inducible promoter present in the mouse mammary tumorvirus long terminal repeat (MMTV LTR) (Klessig et al., Mol. Cell. Biol.,4: 1354 (1984)). Inducible promoters include responsive elements (REs)which stimulate transcription when their inducing factors are bound. Forexample, there are REs for serum factors, steroid hormones, retinoicacid and cyclic AMP. Promoters containing a particular RE can be chosenin order to obtain an inducible response and in some cases, the REitself may be attached to a different promoter, thereby conferringinducibility to the recombinant gene.

Thus, by selecting the appropriate promoter (constitutive versusinducible; strong versus weak), it is possible to control both theexistence and level of expression of a interferon in the geneticallymodified cell. If the gene encoding the interferon is under the controlof an inducible promoter, delivery of the interferon in situ istriggered by exposing the genetically modified cell in situ toconditions permitting transcription of the interferon, e.g., byinjection of specific inducers of the inducible promoters which controltranscription of the agent. For example, in situ expression bygenetically modified cells of interferon protein encoded by aninterferon gene under the control of the metallothionein promoter isenhanced by contacting the genetically modified cells with a solutioncontaining the appropriate (i.e., inducing) metal ions in situ.

Recently, very sophisticated systems have been developed which allowprecise regulation of gene expression by exogenously administered smallmolecules. These include, the FK506/Rapamycin system (Rivera et al.,Nature Medicine 2(9): 1028-1032, 1996); the tetracycline system (Gossenet al., Science 268: 1766-1768,1995), the ecdysone system (No et al.,Proc. Nat. Acad. Sci., USA 93: 3346-3351,1996) and the progesteronesystem (Wang et al., Nature Biotechnology 15: 239-243,1997).

Accordingly, the amount of interferon that is delivered in situ isregulated by controlling such factors as: (1) the nature of the promoterused to direct transcription of the inserted gene, (i.e., whether thepromoter is constitutive or inducible, strong or weak or tissuespecific); (2) the number of copies of the exogenous gene that areinserted into the cell; (3) the number of transduced/transfected cellsthat are administered (e.g., implanted) to the patient; (4) the size ofan implant (e.g., graft or encapsulated expression system) in ex vivomethods; (5) the number of implants in ex vivo methods; (6) the numberof cells transduced/transfected by in vivo administration; (7) thelength of time the transduced/transfected cells or implants are left inplace in both ex vivo and in vivo methods; and (8) the production rateof the interferon by the genetically modified cell.

Selection and optimization of these factors for delivery of atherapeutically effective dose of a particular interferon is deemed tobe within the scope of one of ordinary skill in the art without undueexperimentation, taking into account the above-disclosed factors and theclinical profile of the patient.

Because the protein expressed by our gene therapy methods is a secretedprotein, surrounding cells that do not contain the gene therapy vectorare still affected (see Examples). As a result, the present methodstypically do not require use of a selectable gene. Nevertheless, inaddition to at least one promoter and at least one isolatedpolynucleotide encoding the interferon, the expression vector mayoptionally include a selection gene, for example, a neomycin resistancegene, for facilitating selection of cells that have been transfected ortransduced with the expression vector. Alternatively, the cells aretransfected with two or more expression vectors, at least one vectorcontaining the gene(s) encoding the interferon(s), the other vectorcontaining a selection gene. The selection of a suitable promoter,enhancer, selection gene and/or signal sequence (described below) isdeemed to be within the scope of one of ordinary skill in the artwithout undue experimentation.

IV. Methods of Preparing Specific Gene Therapy Vectors

Any of the methods known in the art for the insertion of polynucleotidesequences into a vector may be used. See, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols inMolecular Biology, J. Wiley & Sons, N.Y. (1992). Conventional vectorsconsist of appropriate transcriptional/translational control signalsoperatively linked to the polynucleotide sequence for a particularinterferon. Promoters/enhancers may also be used to control expressionof interferons. (See Section III)

Expression vectors compatible with mammalian host cells for use in genetherapy of tumor cells include, for example, plasmids; avian, murine andhuman retroviral vectors; adenovirus vectors; herpes viral vectors;parvoviruses; and non-replicative pox viruses. In particular,replication-defective recombinant viruses can be generated in packagingcell lines that produce only replication-defective viruses. See CurrentProtocols in Molecular Biology: Sections 9.10-9.14 (Ausubel et al.,eds.), Greene Publishing Associcates, 1989.

Specific viral vectors for use in gene transfer systems are now wellestablished. See for example: Madzak et al., J. Gen. Virol., 73: 1533-36(1992) (papovavirus SV40); Berkner et al., Curr. Top. Microbiol.Immunol., 158: 39-61 (1992) (adenovirus); Moss et al., Curr. Top.Microbiol. Immunol., 158: 25-38 (1992) (vaccinia virus); Muzyczka, Curr.Top. Microbiol. Immunol., 158: 97-123 (1992) (adeno-associated virus);Margulskee, Curt. Top. Microbiol. Immunol., 158: 67-93 (1992) (herpessimplex virus (HSV) and Epstein-Barr virus (HBV)); Miller, Curr. Top.Microbiol. Immunol., 158:1-24 (1992) (retrovirus); Brandyopadhyay etat., Mol. Cell. Biol., 4: 749-754 (1984) (retrovirus); Miller et al.,Nature, 357: 455-460 (1992) (retrovirus); Anderson, Science, 256:808-813 (1992) (retrovirus).

Preferred vectors are DNA viruses that include adenoviruses (preferablyAd-2 or Ad-5 based vectors), herpes viruses (preferably herpes simplexvirus based vectors), and parvoviruses (preferably “defective” ornon-autonomous parvovirus based vectors, more preferablyadeno-associated virus based vectors, most preferably AAV-2 basedvectors). See, e.g., Ali et al., Gene Therapy 1: 367-384,1994; U.S. Pat.Nos. 4,797,368 and 5,399,346 and discussion below.

The choice of a particular vector system for transferring, for instance,a interferon sequence will depend on a variety of factors. One importantfactor is the nature of the target cell population. Although retroviralvectors have been extensively studied and used in a number of genetherapy applications, they are generally unsuited for infecting cellsthat are not dividing but may be useful in cancer therapy since theyonly integrate and express their genes in replicating cells. They areuseful for ex vivo approaches and are attractive in this regard due totheir stable integration into the target cell genome.

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a therapeutic or reporter transgene to a variety ofcell types. The general adenoviruses types 2 and 5 (Ad2 and Ad5,respectively), which cause respiratory disease in humans, are currentlybeing developed for gene therapy of Duchenne Muscular Dystrophy (DMD)andCystic Fibrosis (CF). Both Ad2 and Ad5 belong to a subclass ofadenovirus that are not associated with human malignancies. Adenovirusvectors are capable of providing extremely high levels of transgenedelivery to virtually all cell types, regardless of the mitotic state.High titers (10¹¹ plaque forming units/ml) of recombinant virus can beeasily generated in 293 cells (an adenovirus-transformed,complementation human embryonic kidney cell line: ATCC CRL1573) andcryo-stored for extended periods without appreciable losses. Theefficiency of this system in delivering a therapeutic transgene in vivothat complements a genetic imbalance has been demonstrated in animalmodels of various disorders. See Y. Watanabe, Atherosclerosis, 36:261-268 (1986); K Tanzawa et al, FEBS letters, 118(1):81-84 (1980); J.L. Golasten et al, New Engl. J. Med., 309 (11983): 288-296 (1983); S.Ishibashi et al, J. Clin. Invest., 92: 883-893 (1993); and S. Ishibashiet al, J. Clin. Invest., 93: 1889-1893 (1994). Indeed, recombinantreplication defective adenovirus encoding a cDNA for the cystic fibrosistransmembrane regulator (CFTR) has been approved for use in severalhuman CF clinical trials. See, e.g., J. Wilson, Nature, 365: 691-692(Oct., 21, 1993). Further support of the safety of recombinantadenoviruses for gene therapy is the extensive experience of liveadenovirus vaccines in human populations.

Human adenoviruses are comprised of a linear, approximately 36 kbdouble-stranded DNA genome, which is divided into 100 map units (m.u.),each of which is 360 bp in length. The DNA contains short invertedterminal repeats (ITR) at each end of the genome that are required forviral DNA replication. The gene products are organized into early (E1through E4) and late (L1 through L5) regions, based on expression beforeor after the initiation of viral DNA synthesis. See, e.g., Horwitz,Virology, 2d edit., ed. B. N. Fields, Raven Press Ltd., New York (1990).

The adenovirus genome undergoes a highly regulated program during itsnormal viral life cycle. See Y. Yang et, al Proc. Natl. Acad. Sci.U.S.A, 91: 4407-4411(1994). Virions are internalized by cells, enter theendosome, and from there the virus enters the cytoplasm and begins tolose its protein coat. The virion DNA migrates to the nucleus, where itretains its extrachromosomal linear structure rather than integratinginto the chromosome. The immediate early genes, E1a and E1b, areexpressed in the nucleus. These early gene products regulate adenoviraltranscription and are required for viral replication and expression of avariety of host genes (which prime the cell for virus production), andare central to the cascade activation of delayed early genes (e.g. E2,E3, and E4) followed by late genes (e.g. L1-L5).

The first-generation recombinant, replication-deficient adenoviruseswhich have been developed for gene therapy contain deletions of theentire E1a and part of the E1b regions. This replication-defective virusis grown in 293 cells which contain a functional adenovirus E1 regionwhich provides in trans E1 proteins, thereby allowing replication ofE1-deleted adenovirus. The resulting virus is capable of infecting manycell types and can express the introduced gene (providing it carries apromoter), but cannot replicate in a cell that does not carry the E1region DNA. Recombinant adenoviruses have the advantage that they have abroad host range, can infect quiescent or terminally differentiatedcells such as neurons, and appear essentially non-oncogenic.Adenoviruses do not appear to integrate into the host genome. Becausethey exist extrachromasomally, the risk of insertional mutagenesis isgreatly reduced. Recombinant adenoviruses produce very high titers, theviral particles are moderately stable, expression levels are high, and awide range of cells can be infected.

Adeno-associated viruses (AAV) have also been employed as vectors forsomatic gene therapy. AAV is a small, single-stranded (ss) DNA viruswith a simple genomic organization (4.7 kb) that makes it an idealsubstrate for genetic engineering. Two open reading frames encode aseries of rep and cap polypeptides. Rep polypeptides (rep78, rep68, rep62 and rep 40) are involved in replication, rescue and integration ofthe AAV genome. The cap proteins (VP 1, VP2 and VP3) form the virioncapsid. Flanking the rep and cap open reading frames at the 5′ and 3′ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp ofwhich are capable of forming Y- or T-shaped duplex structures. Ofimportance for the development of AAV vectors, the entire rep and capdomains can be excised and replaced with a therapeutic or reportertransgene. See B. J. Carter, in Handbook of Parvoviruses, ed., P.Tijsser, CRC Press, pp. 155-168 (1990). It has been shown that the ITRsrepresent the minimal sequence required for replication, rescue,packaging, and integration of the AAV genome.

The AAV life cycle is biphasic, composed of both latent and lyticepisodes. During a latent infection, AAV virions enter a cell as anencapsidated ssDNA, and shortly thereafter are delivered to the nucleuswhere the AAV DNA stably integrates into a host chromosome without theapparent need for host cell division. In the absence of a helper virus,the integrated AAV genome remains latent but capable of being activatedand rescued. The lytic phase of the life cycle begins when a cellharboring an AAV provirus is challenged with a secondary infection by aherpesvirus or adenovirus which encodes helper functions that arerequired by AAV to aid in its excision from host chromatin (B. J.Carter, supra). The infecting parental single-stranded (ss) DNA isexpanded to duplex replicating form (RF) DNAs in a rep dependent manner.The rescued AAV genomes are packaged into preformed protein capsids(icosahedral symmetry approximately 20 nm in diameter) and released asinfectious virions that have packaged either + or − ssDNA genomesfollowing cell lysis.

AAV have significant potential in gene therapy. The viral particles arevery stable and recombinant AAVs (rAAV) have “drug-like” characteristicsin that rAAV can be purified by pelleting or by CsCl gradient banding.They are heat stable and can be lyophilized to a powder and rehydratedto full activity. Their DNA stably integrates into host chromosomes soexpression is long-term. Their host range is broad and AAV causes noknown disease so that the recombinant vectors are non-toxic.

High level gene expression from AAV in mice was shown to persist for atleast 1.5 years. See Xiao, Li and Samuiski (1996) Journal of Virology70, 8089-8108. Since there was no evidence of viral toxicity or acellular host immune response, these limitations of viral gene therapyhave been overcome.

Kaplitt, Leone, Samulski, Xiao, Pfaff, O'Malley and During (1994) NatureGenetics 8, 148-153 described long-term (up to 4 months) expression oftyrosine hydroxylase in the rat brain following direct intracranialinjection using an AAV vector. This is a potential therapy forParkinson's Disease in humans. Expression was highly efficient and thevirus was safe and stable.

Fisher et al. (Nature Medicine (1997) 3, 306-312) reported stable geneexpression in mice following injection into muscle of AAV. Again, thevirus was safe. No cellular or humoral immune response was detectedagainst the virus or the foreign gene product.

Kessler et al. (Proc. Natl. Acad. Sci. USA (1996) 93, 14082-14087)showed high-level expression of the erythropoietin (Epo) gene followingintramuscular injection of AAV in mice. Epo protein was demonstrated tobe present in circulation and an increase in the red blood cell countwas reported, indicative of therapeutic potential. Other work by thisgroup has used AAV expressing the HSV tk gene as a treatment for cancer.High level gene expression in solid tumors has been described.

Recently, recombinant baculovirus, primarily derived from thebaculovirus Autographa califomica multiple nuclear polyhedrosis virus(AcMNPV), has been shown to be capable of transducing mammalian cells invitro. (See Hofmann, C., Sandig, V., Jennings, G., Rudolph, M., Schlag,P., and Strauss, M. (1995), “Efficient gene transfer into humanhepatocytes by baculovirus vectors”, Proc. Natl. Acad. Sci. USA 92,10099-10103; Boyce, F. M. and Bucher, N. L. R. (1996)“Baculovirus-mediated gene transfer into mammalian cells”, Proc. Natl.Acad. Sci. USA 93, 2348-2352).

Recombinant baculovirus has several potential advantages for genetherapy. These include a very large DNA insert capacity, a lack of apreexisting immune response in humans, lack of replication in mammals,lack of toxicity in mammals, lack of expression of viral genes inmammalian cells due to the insect-specificity of the baculovirustranscriptional promoters, and, potentially, a lack of a cytotoxic Tlymphocyte response directed against these viral proteins

IV. Testing for Efficacy/Identification of Interferons

Interferon polynucleotides are administered to a cell via an expressionvector. Generally, one tests the efficacy of a given gene therapy vectoron a particular cellular condition and metabolism by assaying for: (i)alterations in cellular morphology; (ii) inhibition of cellproliferation; and (iii) antiviral activities.

The selection and optimization of a particular expression vector forexpressing a specific interferon gene product in an isolated cell isaccomplished by obtaining the interferon gene, preferably with one ormore appropriate control regions (e.g., promoter); preparing a vectorconstruct comprising the vector into which is inserted the interferongene; transfecting or transducing cultured cells in vitro with thevector construct; and determining whether the interferon gene product ispresent in the cultured cells.

The effect of transfection with polynucleotides encoding interferons maybe tested in vitro using any one of a number of readily available humantumor cell lines. Such cell line include a human bladder carcinoma cellline, 5637 (ATCC HTB9), a human breast carcinoma cell line, MDA-MB468(ATCC HTB132); a human prostate carcinoma cell line, DU145 (ATCC HTB81);a human osteosarcoma cell line, SAOS2 (ATCC HTB85); a human fibrosarcomametastatic to lung cancer cell line, Hs913T (ATCC HTB152); a humancervical carcinoma cell line, HeLa (ATCC ECL 2). Each of these celllines may be transfected with the appropriate polynucleotides encodinginterferons and the effect of transfection on cell growth and cellularmorphology may be tested using procedures known in the art such as theTrypan blue exclusion assay to measure cell viability, cell counting tomeasure propagation over time and tritiated-thymidine incorporation tomeasure DNA replication.

The effect of a secreted protein on surrounding cells that do notcontain a viral vector with the appropriate polynucleotides encoding theprotein, may be easily tested using the methods described in Example 3.

Once introduced into a target cell, interferon sequences can beidentified by conventional methods such as nucleic acid hybridizationusing probes comprising sequences that are homologous/complementary tothe inserted mutant interferon sequences. Interferon transcription canbe measured by reverse transcriptase polymerase chain reaction.Alternatively, interferon protein is measured in the cell-conditionedmedium by conventional antiviral assay or ELISA assay. In anotherapproach, the sequence(s) may be identified by the presence or absenceof a “marker” gene function (e.g, thymidine kinase activity, antibioticresistance, and the like) caused by introduction of the expressionvector into the target cell. For instance, if a polynucleotide encodinginterferon-beta 1a is inserted into a vector having a dominantselectable marker gene such as a neomycin phosphotransferase gene underseparate control of an SV40 early promoter, the sequence can beidentified by the presence of the marker gene function (Geneticinresistance). Other methods of detecting the appropriate vector will bereadily available to persons having ordinary skill in the art.

V. Utilities

A. Interferons and Infectious Diseases

Interferons have been used in the treatment of bacterial, fungal andviral infections. Influenza and vesicular stomatitis virus (VSV) areparticularly sensitive to inhibition by interferons and are often usedin assays to measure interferon activity and in research exploring themechanism of interferon antiviral activity. Other viruses which arehuman pathogens and appear to be sensitive to interferons includehepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus,human papillomavirus, herpes simplex virus, herpes zoster virus,cytomegalovirus (CMV), rhinovirus and encephalomyocarditis virus.

Among the more attractive viral disease targets is hepatitis. Viralhepatitis, a liver disease caused by multiple viruses, is a majorworld-wide health problem. Five distinct human hepatitis viruses havebeen isolated and cloned. These are hepatitis A, B, C, D and E. Somecases of acute and chronic viral hepatitis appear to be associated withhepatitis virus(es) other than those already characterized, such as thenewly discovered hepatitis G virus. Viruses with the highest prevalenceare A, B and C. In addition to causing acute and chronic liver injuryand inflammation, HBV and HCV infection can lead to hepatocellularcarcinoma. Interferons have demonstrated some level of efficacy in vivoagainst HBV and HCV, as well as hepatitis D and hepatitis A in cellculture.

Viral hepatitis could be treated by introduction of the interferon gene.The preferred target for delivery of this gene are the hepatocytes inthe liver. Although ex vivo therapy (e.g., explanting liver cellsfollowed by introduction of the polynucleotide expressing interferon andthen transplantation back into the patient) is possible, gene deliveryin vivo is particularly preferable. Surgery is performed to inject thegene into the portal vein of the liver or the gene is infused through acatheter into the hepatic artery. Ideally, less invasive practices suchas intravenous injection are used.

The interferon gene is placed under the control of a transcriptionalpromoter in a suitable expression vector. The transcriptional promotercan be cellular or viral (CMV, SV40, RSV, etc.) in origin. Ifliver-specific gene expression is desired, a hepatocyte-specificcellular promoter such as the albumin enhancer/promoter, theα1-antitrypsin promoter or an HBV enhancer/promoter is preferred. Thereare many liver-specific promoters described in the literature (Seesupra).

It is also possible to design a vector in which the interferon gene isonly expressed in hepatitis-infected cells. For instance, expression ofthe interferon gene could be induced by the HBV transcription factor HBxwhich will only be present in HBV-infected hepatocytes. In addition, a“carrier” system may be used. This could be a non-viral delivery systemsuch as cationic liposomes or protein:DNA conjugates (conjugates of DNAwith asialoglycoproteins can be delivered preferentially to the livervia binding to the asialoglycoprotein receptor on hepatocytes).

However, to date, the most efficient gene delivery systems are viral inorigin. Recombinant retroviruses have been used to deliver genes tohuman hepatocytes ex vivo. Animal models indicate that recombinantadenovirus can very efficiently localize to the liver followingintravenous in vivo administration. Approximately 98% of adenovirusinjected intravenously localizes to the liver.

It appears that recombinant adeno-associated virus (rAAV) can alsoinfect liver after in vivo administration. Other types of viruses suchas alpha viruses, lentiviruses or other mammalian viruses could be used.Recently, a non-mammalian virus, the insect-specific baculovirus hasbeen shown to be able to deliver and express genes efficiently inhepatocytes (Hofman et al., 1995; Boyce and Bucher, 1996, supra).

By way of example, recombinant adenoviruses can be utilized to deliverthe interferon gene in vivo. Replication-defective adenoviruses havebeen constructed by multiple groups. The first generation of suchviruses are defective due to the deletion of the E1 region. This defectis complemented in the 293 cell line which expresses the adenovirus E1region. It is preferable to use a recombinant adenovirus which has beenmore extensively crippled by deletion of the E1, E2a and/or E4 genes.All of these deleted functions can be expressed in the packaging cellline. The interferon gene is placed downstream of any of a large numberof promoters (for example: the CMV immediate early promoter, the RSVLTR, the cellular actin promoter, the albumin enhancer/promoter or otherliver-specific promoter). This gene cassette is placed into arecombinant adenovirus vector in place of one of the deleted genes, suchas E1 to create the adenovirus transfer vector.

Recombinant adenoviruses having the interferon gene are generated viadirect ligation or homologous recombination following transfection intopackaging cells by standard methods. A recombinant virus stock havingthe interferon gene is plaque purified multiple times then expanded bylarge-scale production in packaging cells. Virus can be purified by CsClbanding, column chromatography or other methods and then titered on thepackaging cells. Methods for generating adenoviruses defective in E1 andE2a (Engelhardt et al., Proc. Nat. Acad. Sci. USA, 91: 6196-6200, 1994)and adenoviruses defective in E1 and E4 (Gao et al., J. Virology, 70:8934-8943, 1996) have been described in detail. Methods for generatingadenoviruses defective in E1 can be found in Graham, F. L. and L.Prevec, “Methods for Construction of Adenovirus Vectors”, Mol. Biotech,3: 207-220 (1995).

Various doses of viruses would be tested in trials. The dose of viruswould likely start at 10⁷ plaque-forming units (pfu) and go up to 10¹²pfu. If necessary, the virus could be administered repeatedly (onceevery one to six months, for instance). A humoral immune responseresulting from repeated viral administration may limit the effectivenessof repeat administration. In this case, a immunosuppressive agent couldbe administered along with the virus such as cyclosporine or antibodydirected against CD40 ligand.

The effect of interferon gene therapy against viral hepatitis can betested in animal models (See Section C). For instance, in the case ofhepatitis B virus, many models are available. These include woodchuck,duck, tree shrew, rat and mouse. Included among the mouse HBV models aremice that are transgenic for HBV and stably replicate HBV DNA in theirlivers leading to the steady production of HBV virus in circulation. ForHCV, a chimpanzee model is available. Adenovirus having an interferongene could be directly administered into liver or be given byintravenous injection. Efficacy would be determined by monitoring viralDNA replication, viral particles in circulation, liver enzymes (ALTs),liver pathology and inflammation.

B. Cancer

Interferon proteins have been shown to possess anti-oncogenic activityin many settings. For reviews, see Wadler and Schwartz, Cancer Research50: 3473-3486, 1990; Martin-Odegard, DN&P, 4: 116-117,1991; and Spiegel,Seminars in Oncology 15 (5): 41-45, 1988. Treatment withinterferon-alpha and interferon-beta have shown some efficacy againstseveral cancers. Gene therapy could be done alone or in conjunction withconventional surgery, radiation or chemotherapy. The following list ofcancers amenable to gene therapy is only a partial one and it is likelythat interferon gene therapy could be effective in a number of diseasesettings which are not included in this list.

Malignant gliomas account for 60-80% of all primary brain tumors inadults. Human glioma cells can be implanted intracerebrally intoimmuno-deficient (nude) mice to provide a glioma model. Interferon-betaprotein treatment has been shown to increase survival in these mice. Aproblem with some of the interferon-beta protein trials in glioma hasbeen the high toxicity following parenteral administration (intravenousor, intramuscular) of interferon-beta. Localized delivery of theinterferon-beta gene into the brain, perhaps at the time of surgery,could result in long-term interferon-beta production in the brainwithout the side-effects seen following systemic protein administration.

Melanoma is an excellent target for interferon gene therapy. Theprognosis for metastatic malignant melanoma is poor. The incidence ofdisease is increasing dramatically and conventional chemotherapies areineffective. Melanoma appears to be an immunogenic tumor type, in thatthe patient response may depend on the host immune response. Both theanti-proliferative and immunomodulatory activities of interferon-betacould be effective in this setting. We have seen that interferon-betaprotein has direct anti-proliferative effects on cultured malignantmelanoma cells.

Hemangioma is a proliferation of capillary endothelium resulting in theaccumulation of mast cells, fibroblasts and macrophages, and leads totissue damage. Although usually harmless, hemangiomas can endanger vitalorgans and cause fatalities. Interferon-alpha protein was shown toinduce early regression of steroid-resistant hemangiomas in infants(Martin-Odegard, supra).

Interferon proteins have been shown to be effective in the treatment ofleukemias, lymphomas and myelomas. The efficacy shown in these diseasesis contrary to the general finding that, although efficacy of interferonproteins in in vitro cancer treatment is well-characterized, in vivoefficacy is far less common. Nevertheless, interferon-alpha isefficacious against hairy cell leukemia, chronic myeloid leukemia,cutaneous T cell lymphomas, Hodgkin's lymphoma and multiple myeloma inhuman clinical trials. Interferon-beta protein inhibits the growth ofrenal cell carcinoma cells in culture. IFN-α has already been approvedfor use in the treatment of renal cell carcinoma.

Colorectal cancer is a major cause of cancer-related deaths in the U.S.There are potent anti-proliferative effects by IFN-α, IFN-β and IFN-γproteins on cultured human colon carcinoma cells. Colon carcinoma oftengenerates metastases in the liver with dire consequences. Adenovirus orother liver-tropic delivery systems could be used to deliver theinterferon gene to the liver for treatment of these metastases.Hepatocellular carcinoma is an attractive target due to the highefficiency of liver delivery by adenovirus. It has been observed thatIFN-β protein significantly inhibits the proliferation of human hepatomacells in culture.

Interferon proteins have shown efficacy in the treatment of inoperablenon-small cell lung carcinoma in some clinical trials, but not inothers. Two human lung cancer cell lines are found to be sensitive togrowth inhibition by interferon-beta protein (beta was more effectivethan alpha). In one clinical trial, significant interferon-relatedtoxicity was observed after intravenous interferon administration. Localdelivery of the interferon-beta gene to the lung (perhaps by aerosoldelivery of a recombinant adenovirus vector) could be efficaciouswithout the toxicity observed following systemic protein delivery. Invitro, the inhibition in proliferation of small-cell lung carcinomacells using interferon-beta protein has been observed.

Interferon-alpha protein inhibits the growth of breast cancer xenograftsin nude mice. Interferon-beta may be efficacious against breast cancerdue not only to its anti-proliferative effects but also due to itsinduction of estrogen receptors and progesterone receptors in vivo tosensitize breast carcinomas to the anti-estrogen tamoxifen. I presentdata from experiments using IFN-β gene therapy in a mouse model of humanbreast carcinoma (See Examples 3 and 4).

Ovarian cancer is a possible disease target. Interferon-beta proteinappears to be less active than interferon-alpha in inhibition ofproliferation of cultured ovarian cancer cells. Therapy, in this case,could be done by installation of the gene therapy vector into theperitoneum as this type of tumor tends to fill the peritoneal cavity.

In summary, interferon proteins have demonstrated anti-oncogenicproperties in a number of settings although clinical results usinginterferon protein are not uniformly positive. IFN-α and IFN-β proteinshave been tested in conjunction with conventional chemotherapeutics andhave shown synergy with these drugs in many indications includingcervical cancer cells, laryngeal carcinoma cells, leukemia cells, renalcell carcinomas, colon adenocarcinoma and myeloma. It is also believedthat interferons possess anti-angiogenesis activity. There is an inversecorrelation between local IFN-β levels and angiogenic capability. Somedata indicate that a sustained level of IFN-β protein is necessary forthe inhibition of angiogenesis. In that case, interferon gene therapywould be preferable to protein therapy in which the high levels ofinterferon protein fall off to low or undetectable levels quite rapidly.

C. Animal Gene Therapy Models

Persons having ordinary skill in the art will be aware of the manyanimal models that are available to test ex vivo and in vivo genetherapy. The most commonly used rodent cancer model is the human tumorxenograft model in nude (nu/nu) mice. The human cancer cells arepropagated in culture and transfected or infected with a gene encodinginterferon operably linked to the appropriate expression controlsequences. These cells are then injected into a nude mouse. Typically,the tumor cells are injected subcutaneously into the back of the mouseleading to the formation of a solid tumor mass (See Example 1).Alternatively, the tumor cells could be injected orthotopically into theorgan in which they would naturally appear (lung cancer cells would beinjected into the lung; colon carcinoma cells into the colon, etc.).Tumor growth can then be followed by measuring the diameter of the tumormass over time (See Example 2).

Okada et al. (1996) Gene Therapy 3, 957-964 formed experimental gliomasin mice by direct intracranial stereotactic injection of human gliomacells into the brain. After detectable tumors formed, an AAV vectorexpressing the herpes simplex virus thymidine kinase gene (HSV tk) wasinjected directly into the same site. The HSV tk enzyme converts thenon-toxic nucleoside analogue gancyclovir (GCV) into a toxic metabolite.After gene therapy, GCV was administered intraperitoneally. Mice whichreceived AAV-tk plus GCV, but not control AAV or AAV-tk without GCV,displayed a dramatic reduction in tumor size. This therapy appeared tobe safe and effective.

Recombinant adenoviruses (AdV) have also been used in the treatment ofsolid tumors in animal models and in early human clinical trials. Manyof these studies used similar nude mouse/human xenograft models. Someexamples of these modeling experiments are listed below. Clayman et al.(1995) Cancer Research 55, 1-6 set up a model of human squamous cellcarcinoma of the head and neck in nude mice. They found that adenovirusexpressing wild type p53 prevented formation of these tumors.

Hirschowitz et al. (1995) Human Gene Therapy 6, 1055-1063 introducedhuman colon carcinoma cells into nude mice. After tumors areestablished, they injected these tumors directly with adenovirusexpressing the E. coli cytosine deaminase gene (CD) then administered5-fluorocytosine (5FC) systemically (CD plus 5FC is a enzyme/pro-drugcombination similar to tk plus GCV). They observed a 4 to 5-foldreduction in tumor size.

Zhang et al. (1996) Proc. Natl. Acad. Sci USA 93, 4513-4518 formed humanbreast tumors in nude mice. These tumors are injected directly withadenovirus expressing interferon-alpha. They observed tumor regressionin 100% of the animals.

Ko et al. (1996) Human Gene Therapy 7, 1683-1691 formed human prostatetumors in nude mice and found that direct intratumoral injection ofadenovirus expressing wild type p53 inhibited tumor growth. All treatedmice remained tumor free for at least 12 weeks after the cessation oftreatment.

Bischoff et al. (1996) Science 274, 373-376 formed human cervicalcarcinoma and glioblastoma tumors in nude mice. They treated these micewith an adenovirus which had a deletion of the E1B gene. In the absenceof E1B, adenovirus selectively kills p53-deficient tumor cells. Wheninjected directly into the tumors, this adenovirus caused tumorregression in 60% of the animals.

Ohwada et al. (1996) Human Gene Therapy 7, 1567-1576 injected humancolon carcinoma cells into the liver of nude mice to mimic livermetastases of colon cancer. They then injected adenovirus expressing CDinto the liver near the tumor. Systemic 5FC treatment suppressed tumorgrowth in these animals.

Cancer models also can be set up in immunocompetent mice and rats. Thesetumors can be established from syngeneic rodent tumor cells which areinjected into the mice. Alternatively, the tumors can derive fromendogenous cells. In these cases, the endogenous tumors could be due totreatment of the animal with a carcinogen or, alternatively, can formspontaneously due to the genetic background of the mouse (deficient inp53, for instance). Some examples follow.

Elshami et al. (1996) Human Gene Therapy 7, 141-148 treated endogenousmesothelioma in immunocompetent rats with adenovirus expressing the tkgene. The virus was injected into the pleural space, and then GCV wasadministered systemically. They showed a dramatic decrease in tumorweight and an increase in survival time.

Eastham et al. (1996) Human Gene Therapy 7, 515-523 implanted syngeneicmouse prostate tumor cell lines subcutaneously into immunocompetentmice. They directly injected adenovirus-tk into the tumor and treatedwith GCV systemically. The authors reported decreased tumor size andprolonged life.

Bramson et al. (1996) Human Gene Therapy 7, 1995-2002 injectedadenovirus expressing the cytokine IL-12 directly into endogenous mousebreast tumors. They found that 75% of the mice had regression of thetumors, and 33% remained tumor free after an extended period of time.

Riley et al. (1996) Nature Medicine 2, 1336-1341 injected adenovirusexpressing the retinoblastoma gene directly into pituitary melanotrophtumors which arose spontaneously in Rb+/− mice. They found decreasedtumor cell proliferation, decreased tumor growth and prolonged life spanin treated animals.

Retrovirus vectors are the first vectors used in human gene therapyclinical trials. One report which is relevant to the present patentapplication is that of Roth et al. (1996) Nature Medicine 2, 985-991.They generated recombinant retrovirus which expressed the wild type p53gene. This virus was introduced into nine human patients havingnon-small cell lung carcinoma by direct intratumoral injection using aneedle inserted in a bronchoscope. Of the nine patients, three displayedtumor regression while three other patients showed stabilization oftumor growth.

D. Other Embodiments

The genetically modified cells are administered by, for example,intraperitoneal injection or by implanting the cells or a graft orcapsule containing the cells in a cell-compatible site of the recipient.As used herein, “cell-compatible site” refers to a structure, cavity orfluid of the recipient into which the genetically modified cell(s), cellgraft, or encapsulated cell expression system can be implanted, withouttriggering adverse physiological consequences. Representativecell-compatible sites include, for example, the peritoneal, pleural andpericardial cavities as well as a solid tumor from which the cells werederived or the organ from which the tumor was removed.

The genetically modified cells are implanted in a cell-compatible site,alone or in combination with other genetically modified cells. Thus, theinstant invention embraces a method for modifying the system of arecipient by using a mixture of genetically modified cells, such that afirst modified cell expresses a first interferon and a second modifiedcell expresses a second interferon or other secreted protein. Othergenetically modified cell types (e.g., hepatocytes, smooth muscle cells,fibroblasts, glial cells, endothelial cells or keratinocytes) can beadded, together with the genetically altered cells, to produceexpression of a complex set of introduced genes. Moreover, more than onerecombinant gene can be introduced into each genetically modified cellon the same or different vectors, thereby allowing the expression ofmultiple interferons by a single cell.

The instant invention further embraces a cell graft. The graft comprisesa plurality of the above-described genetically modified cells attachedto a support that is suitable for implantation into a mammalianrecipient. The support can be formed of a natural or synthetic material.In another embodiment, the graft comprises a patch of peritoneum.Accordingly to this embodiment, the support is the naturally-occurringmatrix that holds the plurality of genetically modified cells together.Alternatively, the graft comprises a plurality of the above-describedcells attached to a substitute for the naturally occurring matrix (e.g.,Gelfoam (Upjohn, Kalamazoo, Mich.), Dacron, Cortex®).

According to another aspect of the invention, an encapsulated cellexpression system is provided. The encapsulated system includes acapsule suitable for implantation into a mammalian recipient and aplurality of the above-described genetically modified mesothelail cellscontained therein. The capsule can be formed of a synthetic ornaturally-occurring material. The formulation of such capsules is knownto one of ordinary skill in the art. In contrast to the cells which aredirectly implanted into the mammalian recipient (i.e., implanted in amanner such that the genetically modified cells are in direct physicalcontact with the cell-compatible site), the encapsulated cells remainisolated (i.e., not in direct physical contact with the site) followingimplantation. Thus, the encapsulated system is not limited to a capsuleincluding genetically-modified non-immortalize cells, but may containgenetically modified immortalized cells.

VI. Formulations.

In a preferred embodiment, the preparation of genetically modified cellscontains an amount of cells sufficient to deliver a therapeuticallyeffective dose of the interferon to the recipient in situ. Thedetermination of a therapeutically effective dose of a specificinterferon for a known condition is within the scope of one of ordinaryskill in the art without the need for undue experimentation. Thus, indetermining the effective dose, one of ordinary skill would consider thecondition of the patient, the severity of the condition, as well as theresults of clinical studies of the specific interferon beingadministered.

If the gene or the genetically modified cells are not already present ina pharmaceutically acceptable carrier they are placed in such a carrierprior to administration to the recipient. Such pharmaceuticallyacceptable carriers include, for example, isotonic saline and otherbuffers as appropriate to the patient and therapy.

The term “pharmaceutically acceptable carrier” means one or moreingredients, natural or synthetic, with which the isolatedpolynucleotide encoding interferon is combined to facilitate itsapplication. A suitable carrier includes sterile saline although otheraqueous and non-aqueous isotonic sterile solutions and sterilesuspensions known to be pharmaceutically acceptable are known to thoseof ordinary skill in the art. In this regard, the term “carrier”encompasses any plasmid and viral expression vectors. An “effectiveamount” refers to that amount which is capable of ameliorating ordelaying progression of the diseased, degenerative or damaged condition.An effective amount can be determined on an individual basis and will bebased, in part, on consideration of the symptoms to be treated andresults sought. An effective amount can be determined by one of ordinaryskill in the art employing such factors and using no more than routineexperimentation.

In preferred methods, an effective amount of the interferonpolynucleotide sequence contained within its attendant vector (i.e.,“carrier”) may be directly administered to a target cell or tumor tissuevia direct injection with a needle or via a catheter or other deliverytube placed into the cancer or tumor or blood vessel feeding the tumor.Dosages will depend primarily on factors such as the condition beingtreated, the selected interferon, the age, weight, and health of thesubject, and may thus vary among subjects. If a viral gene therapyvector is employed, an effective amount for a human subject is believedto be in the range of about 0.1 to about 10 ml of saline solutioncontaining from about 1×10⁷ to about 1×10¹² plaque forming units(pfu)/ml interferon containing, viral expression vectors.

As discussed above, the IFN gene could be administered by directinjection into solid tumors. Alternatively, delivery into the tumorscould be done by infusion into a blood vessel which feeds the tumor.Parenteral administration of the vector is also possible.Polynucleotides encoding interferon may be administered topically,intraocularly, parenterally, intranasally, intratracheally,intrabronchially, intramuscularly, subcutaneously or by any other means.Parenteral administrations will include intravenous, intramuscular,intraperitoneal, and subcutaneous. A more sophisticated approach may beparenteral administration of a virus or chemical conjugate whichlocalizes to a distinct tumor type due to a natural tropism or due tothe presence of a surface molecule which binds to a receptor found onlyon certain tumor types.

As described above, the present invention provides methods for forming acell expression system for expressing a gene product (e.g., ainterferon) in a mammalian recipient, the expression system producedthereby and pharmaceutical compositions containing the same. Thefollowing Examples are directed to demonstrating the feasibility of cellgene therapy in an animal model system

EXAMPLE 1 Exemplary Animal Model

In vivo testing of polynucleotides capable of expressing interferon inan animal model is conveniently accomplished. Tumors are formed in nudemice by injecting human tumor cell lines into the mice. The nude mice(strain nu/nu) are immunodeficient and will not reject the foreign tumorcells. Tumors form 1-2 weeks after tumor cell injection, although theexact timing depends upon the number of cells injected and thetumorigenicity of the cell lines. Polynucleotides expressing interferonare introduced into the tumor cells by conventional transfectionprocedures in culture prior to injection into the mice. Alternately, theappropriate polynucleotide may be introduced into the tumor bytransfection or viral transduction in vivo after the tumors have formedin the mice.

As but one example, the cells used are either the bladder carcinoma cellline HTB9 (Huang et al., supra) or the retinoblastoma cell lineWERI-Rb27 (both Takahashi et al., Proc. Nat. Acad. Sci. USA88:5257-5261, 1991. For delivery of the isolated polynucleotide encodinginterferon in culture, tumor cells are transfected (using a conventionalprocedure such as calcium phosphate precipitation, electroporation, orlipofectamine transfection) or directly infected (using a retrovirus,adenovirus, baculovirus, or adeno-associated virus). In the case of anefficient viral infection in which 100% of cells have successfullyincorporated the appropriate polynucleotide, no selection of cells isrequired. Furthermore, it has been discovered (Example 3) that only asmall percentage of cells need express the interferon gene, so that drugselection usually will not be required.

If selection is performed in the case of transfections which are not asefficient as, the viral infections described herein, cells aretransfected with an expression vector encoding both a drug-resistancegene (such as the neo gene which encodes G418 resistance) and apolynucleotide such as the interferon gene. A control transfection is avector encoding the drug-resistance gene alone.

After about 2-3 weeks of selection in the drug-containing media, thecell colonies are pooled and subcutaneously injected into the flank ofnu/nu (nude) female mice at a cell number of about 10⁶ in a volume ofabout 100 ul. Virus-infected cells are injected directly without theneed for a selection step. The mice are further maintained for at leasttwo months and tumor size is monitored on a weekly basis using calipers.

Alternately, untransfected or uninfected tumor cells are subcutaneouslyinjected in the flank of the mice. After tumor formation, DNA or viruscontaining a polynucleotide encoding an interferon are injected directlyinto the tumors. Many viruses are suitable for this procedure, althoughrecombinant adenoviruses are the most efficient and recombinantretroviruses have the advantage of being stably integrated into thetumor cell genome. DNA can be introduced into the cells by mixing theDNA with cationic liposomes and injecting the mixture. DNA or virusesnot containing the interferon gene are injected into tumors of othermice to serve as the control. Tumor progression or reduction ismonitored with calipers.

EXAMPLE 2 Exemplary Lung Carcinoma Model

As a further example, treatment of human small cell lung carcinoma withliposome-encapsulated, isolated polynucleotide encoding interferon maybe performed in vivo by introducing a polynucleotide encoding interferoninto cells in need of such treatment using liposomes, in particularsmall-particle liposome aerosols. Administered via aerosols,polynucleotide encoding interferon is deposited uniformly on the surfaceof the nasopharynx, the tracheobronchial tree and in the pulmonary area.See, Knight and Gilbert, Eur. J. Clin. Micro. and Infect. Dis., 7:721-731 (1988) for discussion of liposome aerosols. To treat lungcancers in this way, the polynucleotide encoding interferon is purified,by any other convenient method. The polynucleotide encoding interferonis mixed with liposomes and incorporated into them at high efficiency.Since the aerosol delivery is mild and well-tolerated by normalvolunteers and patients, the polynucleotide encodinginterferon-containing liposomes are administered to treat patientssuffering from lung cancers of any stage. The liposomes are delivered bynasal inhalation or by an endotracheal tube connected to a nebulizer ata dose sufficient to inhibit tumor growth. Aerosolization treatments areadministered daily for two weeks, with repetition as needed.

In vivo studies using orthotopic small cell lung carcinoma may becarried out using tumor injected into the right mainstream bronchus ofathymic (nu/nu) nude mice (about 1.5×10⁶ cells per mouse). Three dayslater, the mice begin a course of treatment (daily for three consecutivedays) of being inoculated endobronchially with a liposome-encapsulatedinterferon gene and controls lacking the interferon gene sequences.Tumor formation and size are followed in both treatments by measurmentwith calipers and mouse survival is assessed.

EXAMPLE 3 Ex vivo Gene Therapy with Interferon-beta 1a Gene

In this Example, I use the human breast carcinoma cell line MBA-MD468(obtained from the American Type Culture Collection). Cells are eitheruninfected or infected with an adenovirus expressing the humaninterferon-beta1a gene. In this case, the adenovirus is deleted of theE1 genes and has a temperature sensitive mutation in the E2a gene.Methods of generating this particular adenovirus can be found inEngelhardt et al., (1994), Gene Therapy 5: 1217-1229 (see also below foradditional details). Briefly, the interferon-beta1a gene was previouslycloned into the adenovirus vector pAdCMVlink1 such that genetranscription would be driven by the CMV IE1 promoter, thereby creatingan adenovirus transfer plasmid. The gene was inserted into this vectorin place of the deleted E1 gene. A recombinant adenovirus having thisinterferon gene is generated by recombination of the transfer plasmidand the adenovirus genome in 293 cells. Virus is plaque-purified andtitered in plaque assays by conventional methods.

Materials and Methods

Cell Culture. Human Carcinoma cells MDA-MB468, Huh7, KM12LA4, ME180,HeLa, U87, and 293 are maintained as adherent cultures in Dulbecco'smodified Eagle's. medium containing 10% bovine serum, 2 mM glutamine,penicillin and streptomycin, non-essential amino-acids, and vitamins.

Generation of Purified Adenoviruses. An adenovirus transfer vectorencoding the human IFNIβ gene driven by the cytomegalovirus earlypromoter, pAdCMV-huIFNβ, is constructed by ligating a cDNA insertencoding human IFN-β1a into the plasmid pAdCMVlink1 (see Engelhardt etat, 1994, supra). Plasmid pAdCMV-huIFNβ is co-transferred into 293 cellswith genomic DNA purified from the temperature-sensitive adenovirus H5ts125. Recombinant adenoviruses derived from individual plaques are usedto infect 293 cells at 39° C. and the supematants tested for IFN-β geneexpression by an ELISA assay. An adenovirus carrying the IFN-β cDNA(H5.110 hlIFNβ) is identified and further amplified. Similarly, acontrol E2A temperature-sensitive adenovirus encoding the colorimetricmarker β-galactosidase (H5.1101acZ) is made. Virus preparations areproduced in 293 cells and purified on CsCl gradients after two rounds ofplaque isolation. They were shown to be negative for the presence ofwild-type adenovirus.

Subconfluent cells are infected with H5.110hlIFNβ at multiplicity ofinfection (MOI) of 100 in 3 ml of medium containing 2% bovine serum.Fifteen to eighteen hours later, supematants are collected and IFN-βconcentration quantified by ELISA assay.

ELISA Assay. 96-well plates are coated overnight at 4° C. with ananti-human IFNβ antibody, B02 (Summit Pharmaceuticals Co., Japan). Theantibody is used at 10 μg/ml in the coating buffer containing 50 mMSodium Bicarbonate/carbonate, 0.2 mM MgCl₂, and 0.2 mM CaCl₂ (pH 9.6).After the plates are blocked with 1% casein in PBS for 1 hour at roomtemperature, IFN-β samples of IFN-β protein standards (Avonex™, Biogen),diluted in 1% casein and 0.05% Tween-20, are added. The plates are thensuccessively incubated at room temperature for 1 hour with an anti-IFN-βrabbit sera (1:2000 dilution), 1 hour with horseradish peroxidase(HRP)-conjugated donkey anti-rabbit antibody (Jackson Immuno Research,1:5000 dilution), and the substrate solution (4.2 mM TMB and 0.1 MSodium acetate-citric acid pH4.9). After the reaction is stopped by 2NH₂SO₄, absorbance was measured at 450 nm.

Mouse Experiments. 4 to 6 week old female Balb/c nu/nu mice are obtainedfrom Taconic farms (Boston, Mass.). All mice are maintained in thepathogen-free Biogen animal facility for at least 2 weeks before eachexperiment. For the ex vivo experiments, infected and uninfected cellsare harvested with trypsin/EDTA solution and washed 2 times with PBS.These cells are mixed just prior to injection into mice at the ratiosdescribed below. A total of 2×10⁶ cells in 100 μl of PBS are implantedsubcutaneously into the right flank. Tumor size is measured in lengthand width by using calipers and presented as the average tumor diameter(mm).

For the in vivo direct injection experiments (Example 4), 2×10⁶ tumorcells in 100 μl PBS are first subcutaneously implanted into nude mice.When tumor size reached 5-6 mm in diameter, 100 μl of PBS containingvarious doses of recombinant adenoviruses are injected directly into thecenter of the tumor in a single injection. Tumors are monitored inlength and width using calipers. Tumor size is calculated by averagingthe length and width. Animal death is defined by sacrificing mice inwhich tumors began to show signs of bleeding or reached 10% of totalbody weight. Apoptosis is examined by using the In Situ ApoptosisDetection Kit provided by Oncor, Inc. (Catalog # S7110-KIT).

Results

I initially evaluated the transduction efficiency and transgeneexpression of the adenovirus vectors. Human breast carcinoma cellsMDA-MB-468 are infected with H5.110lacZ at increasing multiplicities ofinfection (MOI). After X-gal staining, I estimated that at an MOI of100, the gene transduction efficiency reached approximately 100% inthese cells (data not shown). Thus, the breast carcinoma cells areinfected in culture at a ratio of 100 plaque forming units (pfu) percell since our experience with these carcinoma cells indicated that thiswas the lowest virus:cell ratio which would lead to expression of thegene in every cell in the population.

For the first experiment, 18 hours after infection, 2×10⁶ cells areinjected subcutaneously into the back of each nude mouse. Five mice areinjected with uninfected cells and five mice are injected with cellsinfected with adenovirus-IFNβ. Tumors of significant size arose in allof the mice injected with the control uninfected cells. No tumorsappeared in any of the mice injected with cells treated with adenovirusexpressing IFN-β (H5.110hIFNβ: Table 1).

To rule out the possibility that in vitro exposure of tumor cells toIFN-β protein might lead to the loss of tumorigenicity in vivo, Itreated MDA-MB-468 cells with IFN-β protein at the protein concentrationthat was detected after the 18 hour virus infection. After thoroughwashing, equal number of treated cells, or untreated cells, or themixture containing 10% treated cells, are injected into the nude mice.Tumor development is observed in all three groups of mice (data notshown), indicating that the ex vivo IFN-β gene delivery, but not invitro protein treatment, is critical to the inhibition of tumorformation.

To determine if cancer cells expressing the IFN-β gene could lead to thedestruction of non-transduced cells, the following experiment isperformed. The same cancer cells are either uninfected, infected withadenovirus expressing the IFN-β gene (H5.110hIFNβ) or infected with thesame type of adenovirus but expressing the lacZ gene (H5.110lacZ) whichencodes the β-galactosidase reporter protein which would not be expectedto have any anticancer effect and, therefore, is a control for anyeffects by the adenovirus itself. All the adenovirus infections are doneat a pfu:cell ratio of 100. I separately infected MDA-MB-468 tumor cellswith H5.110hIFNβ or H5.110lacZ at an MOI at 100. At 18 hours afterinfection, the infected cells were harvested and a portion of them weremixed with uninfected cells just prior to injection into mice. Balb/cnude mice were implanted subcutaneously with equal number of infectedcells, uninfected cells, or a mixture containing 10% infected cells and90% cells which were not exposed to the virus. Tumor growth wasmonitored twelve days later. While all mice implanted with uninfectedcells developed tumors, no tumors are observed in mice that received100% H5.110hIFNβ or H5.110lacZ infected cells, suggesting that infectionby either virus can abolish tumorigenicity (Table 1). However, all micethat received 10% H5.110lacZ infected cells developed tumors, while allmice that received 10% H5.110hIFNβ infected cells failed to do so.Therefore, H5.110lacZ infection, although sufficient to suppress thetumor formation of the already-infected cells, failed to block thetumorigenicity of the co-injected naive and uninfected cells. Incontrast, transduction by H5.110hIFNβ in 10% of cells was enough tosuppress the tumorigenicity of the cells which had been transduced bythe virus as well as those which had not been transduced. Inhibition oftumor formation by H5.110lacZ in the 100% transduced population could bedue to some general toxic effects or to some anti-tumor effects of thisgeneration of adenovirus, but it should be noted that transduced cellswere capable of replication in vitro (unpublished data).

To establish the amount of interferon-containing virus needed tosuppress tumorigenicity, the H5.110hIFNβ and H5.110lacZ infected tumorcells separately are mixed with uninfected cells at various ratios suchthat there was an excess of uninfected cells in each instance. Themixings are such that different samples consisted of 10%, 3%, 1%, 0.3%cells infected with the adenovirus and the remainder uninfected.Immediately after mixing, the cells are injected into the nude mice. Theresults are shown in Table 1. The tumor diameters are measured in twodimensions at various times after injection of the cells. Each datapoint in Table 1 represents the average +/− standard deviation of thelateral and longitudinal diameter measurements from four mice.Measurements are taken at 12, 19, 26 and 33 days after injection of thetumor cells.

TABLE 1 average tumor diameter (in mm) Sample Day 12 Day 19 Day 26 Day33 100% uninfected 4.1 +/− 0.6 4.9 +/− 0.8 5.9 +/− 0.5 6.3 +/− 0.6 100%AdV-IFN 0.0 0.0 0.0 0.0 10% AdV-IFN 0.0 0.0 0.0 0.0 3% AdV-IFN 0.0 0.00.0 0.0 1% AdV-IFN 0.0 0.0 0.0 0.0 0.3% AdV-IFN 2.8 +/− 0.2 2.9 +/− 0.33.0 +/− 0.6 1.8 +/− 2.0 100% AdV-lacZ 0.0 0.0 0.0 0.0 10% AdV-lacZ 4.8+/− 0.4 5.2 +/− 0.6 6.2 +/− 0.5 6.5 +/− 0.5 3% AdV-lacZ 4.3 +/− 0.5 4.6+/− 0.6 5.8 +/− 0.8 6.5 +/− 1.0 1% AdV-lacZ 4.3 +/− 0.4 4.6 +/− 0.4 5.0+/− 0.5 6.3 +/− 0.9 0.3% AdV-lacZ 4.4 +/− 0.5 4.7 +/− 0.6 ND ND

Tumor development was completely blocked in mice that received as few as1% H5.110hIFNβ transduced cells (Table 1). In the first week afterinjection of the cells, very small tumors could be detected (these arepalpable, but not big enough to be measured) in the mice injected withthe 10, 3 and 1% H5.110hIFNβ However, all of these small tumorscompletely regressed by day 9. This suggests that some cells survivedfor a short time and expressed the IFN-β gene during this period,leading to death of the entire tumor. In the several experimentsperformed with this cell line in nude mice, tumor formation has neverbeen observed when 1% of cells were transduced with H5.110hIFNβ andsurvival has been 100% (data not shown). Mice that received 0.3%H5.110hIFNβ transduced cells developed tumors, however, the size ofthese tumors was significantly smaller than those in the control miceand 2 out of 5 mice in this 0.3% group had complete regression by day 33(Table 1).

In contrast, mice that receive 10% to 0.3% H5.110lacZ treated cellsdevelop tumors with similar sizes as the uninfected group and no tumorregression was observed in either of these control groups (Table 1).

Clearly, expression of human interferon beta (hIFN-β) in only a verysmall percentage cells appeared to block tumorigenesis in nude mice. Ifurther examined the lowest ratio for H5.110hlIFNβ infected cellsrequired to affect, but not necessarily block, tumor formation andpromote mouse survival. Equal numbers of MDA-MB-468 cells containing0.3%, 0.1%, 0.03%, 0.01%, and 0% H5.110hIFNβ infected cells areimplanted into nude mice and tumor growth was monitored. Mice thatreceive 0.3% or 0.1% infected cells develop much smaller tumors comparedwith those that received only uninfected cells (FIG. 1A). Of the tumorswhich formed at 0.3 and 0.1% transduction, 3 out of 5 and 1 out of 5tumors, respectively, regressed completely. Significantly prolongedsurvival was observed in the 0.3% and 0.1% transduction groups. Whileimplantation of 0%, 0.01%, or 0.03% infected cells resulted in the deathof all animals within 75 days, 1 out of 5 and 3 out of 5 animals werealive without tumors in the 0.1% and 0.3% groups, respectively, at theconclusion of this experiment on day 109 (FIG. 1B).

It is likely that only a small portion (greater than about 0.3%,preferably greater than about 1.0% ) of all the tumor cells will need tobe transfected or infected in order to have efficacy. This differs fromsuch anticancer gene therapy approaches such as delivery of wild typetumor suppressors (p53, for example) in which every cell in the tumorwill need to obtain the tumor suppressor gene.

Thus, the interferon gene demonstrates a potent anti-proliferativeeffect in vivo after in vitro infection. Controls indicate that this wasdue to interferon rather than the adenovirus.

Ex vivo IFN-β Gene Therapy in Other Human Xenograft Tumors

I also tested the effect of H5.110hIFNβ transduction in other tumor celltypes in the ex vivo human xenograft model. Human colon carcinoma cellsKM12L4A, human liver carcinoma cells Huh7, or human cervical carcinomacells ME180 are transduced with H5.110hIFNβ Equal number of cellscontaining 10%, 1%, or 0% transduced cells are tested for their abilityto form tumors in nude mice. Injection of uninfected cells of the threetypes causes the formation of fast growing tumors in all mice. Incontrast, ex vivo delivery of 10% H5.110hIFNβ infected cells leads toeither no tumor development or the delayed appearance of slower growingtumors in all animals examined (FIG. 2A). Unlike results obtained withMDA-MB-468 cells in which 1% transduction by H5.110hIFNβ completelyprevented tumor formation, 1% transduction of these three cell typesresults in the formation of tumors, although their sizes are smallerthan the uninfected controls at each time point. Mice that received 10%and 1% transduced cells exhibit prolonged survival compared to thosethat received uninfected cells (FIG. 2B). Thus, ex vivoadenovirus-mediated IFN-β gene delivery into multiple different tumorcells results in efficient inhibition of tumorgenicity and leads toincreased animal survival time.

EXAMPLE 4

In vivo Gene Therapy with Interferon-beta 1a Gene

Instead of an ex vivo approach direct in vivo gene therapy can beperformed. In in vivo gene therapy, the gene is directly administeredinto the patient. In this Example, adenovirus having the human IFN-βgene (H5.110hIFNβ is directly injected into solid tumors. Briefly, 2×10⁶MBA-MD-468 human breast carcinoma cells are injected subcutaneously intothe back of fifty nude mice. Subcutaneous tumors of approximately 5-6 mmdiameter form in nude mice 24 days following subcutaneous injection ofMDA-MB-468 ceus. At this time, tumors were treated with PBS or theH5.110hIFNβ and H5.110lacZ vectors at various viral doses ranging from1×10⁷ to 3×10⁹ pfu/mouse in a single intratumor injection.

Data shown in FIG. 3 indicate that within 14 days, single dose treatmentwith H5.110hIFNβ at 3×10⁹, 1×10⁹, or 3×10⁸ total pfu causes tumorregression. Complete tumor regression occurs in 4 out of 5 mice in the3×10⁹ pfu group and in 3 out of 5 animals in the 1×10⁹ pfu treaunentgroup. In tumors injected with 1×10⁹ pfu H5.110hIFNβ a high local IFN-βconcentration of approximately 1500 IU/ml can be detected while only 37IU/ml of INF-β is detected in the seruml Lower H5.110hIFNβ dosesincluding 1×10⁸, 3×10⁷ and 1×10⁷ pfu have little or no effect (FIG. 3and data not shown), indicating that the anti-tumor response isdose-dependent. Injection of PBS or H5.110lacZ at equivalent doses doesnot lead to tumor regression (FIG. 3.) When the tumors are monitoredover a longer period of time, slow growth and regression are observed insome individual tumors injected with H5.110lacZ at 3×10⁹ pfu, suggestingthat the control virus at that dose may cause slight inhibition of tumorgrowth. Treatment with H5.110hIFNβ at 3×10⁹, 1×10⁹, or 3×10⁸ pfusignificantly increases survival relative to PBS or H5.110lacZ treatedmice (data not shown). We have also tested multiple injections, with 5injections of 1×10⁸ pfu H5.110hIFNβ given every third day intoestablished MDA-MB-468 and HeLa tumors resulting in slower growth andregression of both tumor types (unpublished). I also performed a similarin vivo experiment using the human glioma cell line U87. These cellswere very sensitive to IFN-β as complete tumor regression was seen in 4out of 4 mice treated with 1×10⁹ pfu H5.110hIFNβ and 2 out of 4 micetreated with 1×10⁸ pfu (data not shown). These findings demonstrate thatdirect and local in vivo adenovirus delivery of the IFN-β gene can exerta significant anti-tumor effect.

Four days after injection with 1×10⁹ pfu virus, the MDA-MB-468 tumorsare harvested for histological examination. At that time, tumorsinjected with H5.110hIFNβ show signs of regression. Histologicalanalysis of the MIDA-MB-468 tumors by hematoxylin-eosin staining isperformed. More apoptotic cells are noted in the H5.110hIFNβ-injectedtumor than in the H5.1101acZ-injected tumor. I confirmed apoptosis bydirect fluorescence detection of end-labeled, fragmented genomic DNA.Very few infiltrating mononuclear cells are observed in the H5.110hIFNβor HS.1101acZ injected tumors, indicating that the cellular immuneresponse may not play a major role in the H5.110hIFNβ directed tumorregression in this model.

Both the ex vivo and in vivo experiments shown above measure only thedirect anti-proliferative effect of interferon. Since these areimrnmune-deficient mice, any immuno-stimulatory activity that interferonhas which might stimulate tumor destruction will not be present. Also,since interferons do not cross species from human to mouse, the humanIFN-β used to inhibit the human cancer cells in these mice would not beexpected to inhibit angiogenesis since the human IFN-β does not act onthe mouse vascular endothelial cells. Therefore, it is possible that aneven more dramatic anti-cancer activity would be seen when humanpatients are treated with the adenovirus having the human IFN-β. Thiscould be modeled in immune-competent non-human primates. Alternatively,one could use adenovirus having the murine IFN-β gene inimmune-competent mice having tumors of murine origins. Many of thesesyngeneic mouse tumor models are available.

The data provided here demonstrate a remarkable ability of IFN-β genetherapy to block the formation of tumors de novo as well as to causeregression of established tumors. The ex vivo transdpctions experimentsconfirmed that introduction of a potent secreted protein into as few as0.3%-1.0% transduced cells blocked the establishment of MDA-MB-468tumors. A variety of other tumor cell lines have been tested, and whilethere was a variation in the potency of the IFN-β effect, all could beblocked with 1-10% of IFN-β transduced cells. Encouraged by therelatively small percentage of IFN-β secreting cells required to impacttumor formation, I then challenged pre-formed tumors with directintratumor injection of the adenoviruses. Again the effect of the IFN-βgene delivery was potent with single injections of virus resulting ineither partial or in some cases complete regression of tumors.

In these studies, the dramatic regression of tumors appeared to beprimarily the result of the direct anti-proliferative or cytotoxicactivity of IFN-β. This conclusion is supported by the fact that theIFN-β gene used in this study is of human origin, and human IFN-β doesnot cross react appreciably with the host mouse cells. Also, theimmune-deficient nude mice that were used lack T lymphocytes, a majoreffector cell in the type 1 IFN induced immunostimulation (Tough, D. F.et al., (1996), Science 272: 1947-1950 and Rogge, L. et al., (1997) J.Exp. Med., 185: 825-831). Furthermore, in the rapidly regressing tumorsfollowing IFN-β gene delivery, no overt increase in the infiltration ofmononuclear cells was observed. These findings support the notion thatIFN-β mediated anti-proliferative activity alone could be sufficient tocause tumor regression. Our data appear to be consistent with theclinical correlation previously observed between the in vitrosensitivity of malignant cells to IFN-induced anti-proliferativeactivity and the in vivo therapeutic effect (Einhorn and Grander (1996)J. Interferon Cytokine Res. 16: 275-281)

In summary, it has been found that adenovirus-mediated IFN-β genetherapy can exert an efficient anti-tumor effect in mouse models. Exvivo delivery of the IFN-β gene into a very small percentage of cellswas sufficient to block tumor formation and single-dose directintra-tumor IFN-β gene delivery led to regression of established tumors.Without wishing to be bound by any theory of actions, this potentanti-tumor effect, may result from autocrine and paracrine effect ofIFN-β. This anti-tumor effect could be a critical factor in gene therapycancer trials in which the degree of gene delivery is likely to belimiting and a significant bystander effect will be required. Therefore,local IFN-β gene therapy provides a promising strategy for the treatmentof tumors in humans.

Equivalents

It should be understood that the preceding is merely a detaileddescription of certain preferred embodiments. It therefore should beapparent to those skilled in the art that various modifications andequivalents can be made without departing from the spirit or scope ofthe invention.

1. A method for treating cancer by in vivo interferon-β gene therapycomprising the steps of: parenterally administering to a subject a viralvector comprising a gene that encodes interferon-β protein, and allowingsaid interferon-β protein to be expressed from said gene in said subjectin an amount sufficient to cause cancer regression or inhibition ofcancer growth, wherein said viral vector is selected from the groupconsisting of an adenoviral vector, a lentiviral vector, a baculoviralvector, an Epstein Barr viral vector, a papovaviral vector, a vacciniaviral vector, and a herpes simplex viral vector, and wherein if saidviral vector is an adenoviral vector, then said subject is not exposedto a nucleic acid encoding a selectable marker gene.
 2. The methodaccording to claim 1, wherein said viral vector is selected from thegroup consisting of an adenoviral vector, a baculoviral vector and alentiviral vector.
 3. The method according to claim 1, wherein saidviral vector is an adenoviral vector.
 4. The method according to claim3, wherein said adenoviral vector has a deletion in its E3 gene.
 5. Themethod according to claim 3, wherein said adenoviral vector has adeletion in its E1 gene.
 6. The method according to claim 4, whereinsaid adenoviral vector has a deletion in its E1 gene.
 7. The methodaccording to claim 1, wherein said parenteral administration is selectedfrom the group consisting of intravenous administration, intramuscularadministration, subcutaneous administration and intraperitonealadministration.
 8. The method according to claim 1, wherein said canceris selected from the group consisting of malignant glioma, melanoma,hemangioma, leukemia, lymphoma, myeloma, colorectal cancer, non-smallcell carcinoma, breast cancer and ovarian cancer.
 9. The methodaccording to claim 8, wherein said cancer is malignant glioma.
 10. Themethod according to claim 1, wherein said gene encodes a humaninterferon-β protein.
 11. The method according to claim 1, wherein saidsubject is a human subject.
 12. A method for treating cancer by in vivointerferon-β gene therapy comprising the steps of: parenterallyadministering to a subject a replication-defective viral vectorcomprising a gene that encodes interferon-β protein, and allowing saidinterferon-β protein to be expressed from said gene in said subject inan amount sufficient to cause cancer regression or inhibition of cancergrowth, wherein said viral vector is selected from the group consistingof an adenoviral vector, a lentiviral vector, a baculoviral vector, anEpstein Barr viral vector, a papovaviral vector, a vaccinia viral vectorand a herpes simplex viral vector, and wherein if said viral vector isan adenoviral vector, then said subject is not exposed to a nucleic acidencoding a selectable marker gene.
 13. The method according to claim 12,wherein said viral vector is selected from the group consisting of anadenoviral vector, a baculoviral vector and a lentiviral vector.
 14. Themethod according to claim 12, wherein said viral vector is an adenoviralvector.
 15. The method according to claim 14, wherein said adenoviralvector is deficient is one or more essential genes of one or moreadenoviral genome regions selected from the group consisting of the E1,E2A and E4 regions of the adenoviral genome.
 16. The method according toclaim 14, wherein said adenoviral vector has a deletion in its E3 gene.17. The method according to claim 14, wherein said adenoviral vector hasa deletion in its E1 gene.
 18. The method according to claim 17, whereinsaid adenoviral vector is deficient in its E3 gene.
 19. The methodaccording to claim 12, wherein said parenteral administration isselected from the group consisting of intravenous administration,intramuscular administration, subcutaneous administration andintraperitoneal administration.
 20. The method according to claim 12,wherein said cancer is selected from the group consisting of malignantglioma, melanoma, hemangioma, leukemia, lymphoma, myeloma, colorectalcancer, non-small cell carcinoma, breast cancer and ovarian cancer. 21.The method according to claim 20, wherein said cancer is malignantglioma.
 22. The method according to claim 12, wherein said gene encodesa human interferon-β protein.
 23. The method according to claim 12,wherein said subject is a human subject.
 24. A method for treatingcancer by in vivo interferon-β gene therapy comprising the steps of:administering to a subject a viral vector comprising a gene that encodesinterferon-β protein, and allowing said interferon-β protein to beexpressed from said gene in said subject in an amount sufficient tocause cancer regression or inhibition of cancer growth by induction of asystemic anti-tumor immune response, wherein said viral vector isselected from the group consisting of an adenoviral vector, a lentiviralvector, a baculoviral vector, an Epstein Barr viral vector, apapovaviral vector, a vaccinia viral vector and a herpes simplex viralvector, and wherein if said viral vector is an adenoviral vector, thensaid subject is not exposed to a nucleic acid encoding a selectablemarker gene.
 25. The method according to claim 24, wherein said viralvector is selected from the group consisting of an adenoviral vector, abaculoviral vector and a lentiviral vector.
 26. The method according toclaim 24, wherein said viral vector is an adenoviral vector.
 27. Themethod according to claim 26, wherein said adenoviral vector has adeletion in its E3 gene.
 28. The method according to claim 26, whereinsaid adenoviral vector has a deletion in its E1 gene.
 29. The methodaccording to claim 27, wherein said adenoviral vector has a deletion inits E1 gene.
 30. The method according to claim 24, wherein said geneencodes a human interferon-β protein.
 31. The method according to claim24, wherein said subject is a human subject.